Nuclear magnetic resonance assembly of chemical entities using advanced antenna probes

ABSTRACT

The invention provides a method for identifying a ligand that binds to a macromolecular target. The methods involve (a) attaching an antenna moiety to a first ligand, wherein the ligand binds specifically to a macromolecular target; (b) providing a sample comprising the macromolecular target, the first ligand and a candidate second ligand under conditions wherein the first ligand and the macromolecular target form a bound complex; (c) detecting a subset of magnetization transfer signals between the antenna moiety of the first ligand and the second candidate ligand, wherein the signals are obtained from an isotope edited NOESY spectrum of the sample; thereby determining that the two ligands are proximal in a bound complex, and identifying a second ligand that binds to the macromolecular target.

This application claims the benefit of U.S. Provisional Application No.60/455,610, filed Mar. 13, 2003, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

The present invention relates generally to drug discovery methods and,more specifically to Nuclear Magnetic Resonance (NMR) methods foridentifying compounds that interact with macromolecules.

Two general approaches have traditionally been used for drug discovery:structure-based drug design and screening for lead compounds.Structure-based drug design utilizes a three-dimensional structure modelof a drug target to predict or simulate interactions with known orhypothetical compounds. Alternatively, in cases where athree-dimensional structure model of a drug target complexed with aligand is available, therapeutic drugs can be designed to mimic thestructural properties of the ligand, thereby identifying lead compoundsfor further development.

Screening for lead compounds is another approach that has been used withsome success to identify lead compounds for therapeutic targets.Screening involves assaying a library of candidate compounds to identifylead compounds that interact with a drug target. The probability ofidentifying a lead compound can be increased by providing increasednumbers and variety of candidate compounds in the library to bescreened. Synthetic methods are available for creating libraries ofcompounds and include, for example, combinatorial chemistry approachesin which selected chemical groups are variously combined to generate alibrary of candidate compounds having diverse combinations of theselected chemical groups. In addition, advances have been made toincrease the throughput for a number of screening methods. However, formany drug targets the throughput of available screens is prohibitivelylow. Furthermore, even in cases where high throughput detection isavailable, limitations on available resources for obtaining a librarywith sufficient size or diversity, or for obtaining a sufficientquantity of the drug target to support a large screen, can beprohibitive.

The efficiency of library screening approaches can be increased bycombining structure-based drug design with the methodologies currentlyavailable for library screening. In particular, the probability ofidentifying a lead compound in a screening approach can be increased byusing focused libraries containing member compounds having a higherprobability of interacting with the drug target. Focused librarieshaving members with a limited range of structural or functionalvariations have been obtained based on variations predicted fromstructure-based drug design methods and used to screen for candidatedrugs.

However, for many drug targets of interest, three-dimensional structuremodels are not presently available. Although methods for structuredetermination are evolving, it is currently difficult, costly and timeconsuming to determine the structure of a macromolecule drug target atsufficient resolution to render structure-based drug design practical.It can often be even more difficult to produce a macromolecule-ligandcomplex in a condition allowing a sufficiently resolved structure modelof the complex. The typically long time period required to obtainstructure information useful for developing drug candidates isparticularly limiting with regard to exploiting the growing number ofpotential drug targets identified by genomics research.

Thus, there exists a need for methods to reduce the size and diversityof candidate libraries required to screen for lead compounds. Thepresent invention satisfies this need and provides related advantages aswell.

SUMMARY OF THE INVENTION

The invention provides a method for identifying a ligand that binds to amacromolecular target. The methods involve (a) attaching an antennamoiety to a first ligand, wherein the ligand binds specifically to amacromolecular target; (b) providing a sample comprising themacromolecular target, the first ligand and a candidate second ligandunder conditions wherein the first ligand and the macromolecular targetform a bound complex; (c) detecting a subset of magnetization transfersignals between the antenna moiety of the first ligand and the secondcandidate ligand, wherein the signals are obtained from an isotopeedited NOESY spectrum of the sample; thereby determining that the twoligands are proximal in a bound complex, and identifying a second ligandthat binds to the macromolecular target.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of a method for sequentially building abinding compound from three ligands.

FIG. 2 shows structures for ligands that are proximal to PBBA when boundto p38α MAP kinase, where the PBBA terminal methyl is represented by anasterisk in part b and atoms that have NOE interactions are indicated byarrows.

FIG. 3 shows NMR NOESY spectra for PBBA and inhibitor TTM2001.082.B09when bound to p38α (left panel) and structures for PBBA and inhibitorTTM2001.082.B09 (right panel).

FIG. 4 shows the structure of the TTM2001.101.A09 bi-ligand and ¹H NMRspectra for the TTM2001.101.A09 bi-ligand in the absence (a) andpresence (b) of 10 uM p38α MAP kinase.

FIG. 5 shows IC₅₀ values for inhibition of myelin basic proteinphosphorylation by p38α MAP kinase in the presence of inhibitorTTM2001.082.B09, PBBA or TTM2001.101.A09, respectively.

FIG. 6 shows structures for PBBA analogs that bind to p38α MAP kinase.Atoms that have NOE interactions with TTM2001.070.A10 are indicated byarrows.

FIG. 7 shows NMR NOESY spectra for PBBA and SB203580 when bound to p38αand structures for PBBA and SB203580 (upper panel) and acrystallographic structure model of the SB203580/p38α complex where thePBBA binding region is indicated by the white oval (lower panel).

FIG. 8 shows a schematic diagram of the relative locations where ATP(dark circle), myelin basic protein (white circle), peptide (greycircle) and PBBA (area within the white circle and indicated bybrackets) bind to p38α MAP kinase.

FIG. 9 shows a structure model of p38α derived from Wang et al.Structure 6:1117-1128 (1998) in which residues are colored to indicatehomology within the family of p38α-like proteins. The pentagon indicatesthe location of inhibitor SB203580 binding and the white circleindicates the location of PBBA binding, both determined from dockingsimulations.

FIG. 10 shows the structures of TTM2002.143.A27 and TTE0020.003.A05 withNOE interactions indicated by arrows.

FIG. 11 shows the structures of TTE0020.003.A09 and TTM2002.143.A27 withNOE interactions indicated by arrows.

FIG. 12 shows the structures of TTM2002.143.A27 and TTE0020.002.H10 withNOE interactions indicated by arrows.

FIG. 13 shows a synthetic scheme for labeling a ligand with an antennamoiety.

FIG. 14 shows a synthetic scheme for labeling a ligand with a C¹³/H²antenna moiety.

FIG. 15 shows structures for a portion of one ligand proximal to aportion of a ligand-probe when both ligand and ligand-probe are bound top38α, and the corresponding ¹³C-edited NOESY spectra with NOEinteractions indicated by arrows.

FIG. 16 shows NOESY spectra for the ligand and ligand-probe shown inFIG. 15 with NOE interactions indicated by arrows, obtained using a 1hour acquisition time (A) or 4 hour acquisition time (B).

FIG. 17 shows 1D gradient sculpted NOESY spectra for the ligand andligand-probe shown in FIG. 15 with NOE interactions indicated by arrows.

FIG. 18 shows a representation of the distances between a specificityligand fragment (F1) antenna moiety and a common ligand fragment (F2).

FIG. 19 shows NOESY spectra for a portion of a ligand and inhibitorTTM2002.143.A27, with NOE interactions indicated by arrows.

FIG. 20 shows NOESY spectra for a portion of a ligand and inhibitorTTM2002.143.A27, when the antenna moiety is deuterated, with NOEinteractions indicated by arrows.

FIG. 21 shows a representation of the binding of a ligand-probe to amolecule, whether the common ligand portion and specific ligand portionare linked (A), or separated (B). FIG. 21C shows a representation of thebinding of a common ligand portion of a ligand-probe (F2 fragment) and aproximal specificity ligand moiety (F1 fragment) to a substrate FIG. 21Dshows a representation of the binding of a common ligand portion of aligand-probe and a proximal ligand moiety (F2 fragment) to a substrate.

FIG. 22 shows the structure of a 4-fluoro-piridyl-pyrazole core ATPmimic and a 4-chlorophenol lead fragment with a reverse NMR ACE ureaantenna moiety, and the corresponding 2D IL-NOESY spectrum obtained withthese compounds in the presence of p38α.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for identifying a compound that willbind to a macromolecule. Using a method of the invention, the relativepositions of two or more ligands when bound to a macromolecule in amultipartite complex can be determined. Based on this determination, theligands or portions of the ligands can be covalently linked to form abinding compound. An advantage of the invention is thatatomic-resolution structural data for a macromolecule, although usefulin some aspects of the invention, is not necessary in order to obtain acompound that binds to the macromolecule.

The methods can be used to design a compound that binds to a particularmacromolecular target. The compound can be designed by determining therelative positions of two or more ligands when bound to a macromoleculartarget in a multipartite complex. The methods involve attaching anantenna moiety to a known ligand (to produce a ligand-probe), incubatingthe macromolecular target; ligand-probe and a candidate ligand underconditions wherein the ligand-probe and macromolecular target form abound complex; and detecting whether the ligand-probe and candidateligand are in proximity to each other. In addition, the distance betweenthe antenna moiety of the ligand-probe and candidate ligand andorientation of a candidate ligand with respect to a ligand-probe can bedetermined.

The methods can also be used to design a library that is focused towardmembers of a particular protein family having a common ligand site(CLS). The focused library can include compounds with variouscombinations of linked moieties, where the moieties are structurallysimilar to each ligand observed in a multipartite complex and the linkerbetween the moieties is selected based on the relative positions of theligands in the multipartite complex. A focused library can be designedby determining the relative positions of two or more ligands when boundto a macromolecule in a multipartite complex, identifying which of theligands is a common ligand capable of binding to the CLS and buildingthe library to contain members having the common ligand linked tovarious moieties that are structurally similar to the other ligand. Anadvantage of the invention is that screening with one or arepresentative subset of proteins in a family can be used to design alibrary that is focused with respect to other proteins in the family.

Nuclear Magnetic Resonance (NMR) can be used in a method of theinvention to determine the relative proximity or positions of ligands ina multipartite complex with a macromolecule. In particular, proximalligands can be identified from NMR-based observation of magnetizationtransfer between the ligands. Although NMR methods have been previouslyused to predict or determine the structure of ligands bound tomacromolecules, these methods have relied upon detection of magneticinteractions between the ligand and the macromolecule. Isotopic labelingcan be required for macromolecules in order to detect magneticinteractions with a bound ligand. Furthermore, for many large ormembrane bound macromolecules signal broadening, due in part to lowrotational mobility, renders detection of magnetic interactions withligands impractical. Because the methods of the present invention arebased on detection of interactions between ligands and do not requiredetection of interactions with macromolecule, isotopically labeledmacromolecules are not necessary. The methods are further advantageousfor use with large or membrane bound macromolecules because observationof magnetization transfer between ligands can be enhanced when theligands experience low rotational mobility.

In several embodiments, the methods of the invention advantageouslyinvolve using an isotope filter to select or reject magnetization thatoriginates solely from a portion of a ligand molecule, such as a ligandcore structure or antenna moiety. Thus, the resulting spectra contain areduced number of resonances. The application of isotope-editing orfiltering of NMR experiments in the methods of the invention allows forreduced data complexity and a corresponding simplified data analysis.Simplified NMR data analysis in turn allows for faster data analysis, aswell as the reduced training time required for individuals to interpretexperimental data. Moreover, isotope-edited or filtered NMR experiments,such as those described in Example VII, can be performed in short timeframes that make feasible high sample throughput.

A further advantage of the invention is that ligands having relativelylow affinities for a macromolecule can be identified and linked to forma compound having substantially increased affinity for themacromolecule. Such increased affinity is expected to occur, forexample, due to the chelate effect (for a description of the chelateeffect see Page et al., Proc. Natl. Acad. Sci. USA 68:1678-1683 (1971))and is demonstrated in the Examples below. Another advantage of theinvention is that a compound having increased specificity for aparticular macromolecule, compared to a ligand from which it isassembled, can be identified. In particular, members of a CLS-containingprotein family often have a different specificity ligand site adjacentto the common ligand binding site which provides a potential source ofbinding specificity (as described, for example, in U.S. patentapplication Ser. No. 09/328,322 and WO 99/60404). By linking the commonligand to a particular specificity ligand, a compound can be obtainedthat has increased affinity due to the presence of both ligands andincreased specificity for a particular member of a protein familycompared to the common ligand.

Yet another advantage of the invention is that once a specificity ligandis identified, it can be used in methods for identifying other proximalligands, including other common or specificity ligands. The methods ofthe invention further can be used to refine, or optimize, an identifiedligand to select a ligand with increased or decreased proximity toanother ligand, with respect to the originally identified ligand.

A ligand can include an antenna moiety that extends from the corestructure of the ligand to interact with a proximal ligand. An antennamoiety can extend the range within which a proximal ligand isidentified. The use of a multi-probe ligand having multiple antennamoieties or comparison of ligands having antennas of differentcomposition or point of attachment on the ligand moiety can provideinformation on the relative orientation of the proximal ligands andtheir binding sites. In a method of the invention that employsisotope-edited NOESY, an antenna moiety can contain one or moreNMR-visible nuclei such as ¹³C, ¹⁵N, ¹⁹F, ³¹P, ¹¹³Cd, and the like. Anantenna moiety also can contain NMR-invisible nuclei, such as ²H, forsome applications.

In one embodiment, the invention provides a compound comprising amolecule attached to an antenna moiety that contains both a ¹³C nucleusand several ²H nuclei.

Because the methods of the invention provide not only a functionalidentification that a ligand binds to a macromolecule, but also identifythe relative positions of two ligands when bound to the macromolecule,the invention provides structural information. Use of a method of theinvention in a screening format provides a way to increase thethroughput at which structural information can be obtained on therelative orientation of the proximal ligands and their binding sites.

In the following description, for the purposes of explanation, specificdetails are set forth in order to provide a thorough understanding ofthe present invention. Those skilled in the art will understand that thepresent invention can be practiced without these specific details andcan be applied to any of a variety of related systems. For example,although the methods are described in the context of ligands that bindto a protein, it is understood that the methods can be applied to othermacromolecules including, for example, synthetic polymers, DNA, RNA orpolysaccharides that interact with ligands.

As used herein, the term “ligand” is intended to mean a molecule thatcan form a specific, non-covalent association with a macromolecule. Amolecule included in the term can be a small molecule, a bindingcompound or a macromolecule. A molecule included in the term can benaturally occurring such as a DNA, RNA, polypeptide, protein, lipid,carbohydrate, amino acid, nucleotide, metabolite or hormone; a syntheticmolecule; or a derivative of a naturally occurring molecule. Aderivative can have, for example, an added moiety, a removed moiety or arearrangement in the relative location of moieties compared to anaturally occurring molecule. As used herein, the term “bindingcompound” is intended to mean a ligand having a covalent structure thatincludes at least two moieties that interact with a macromolecule.

As used herein, the term “binding site” is intended to mean a portion ofa macromolecule or complex of macromolecules that associatesspecifically and non-covalently with a ligand or portion of a ligand. Anon-covalent association included in the term can be due to a hydrogenbond, ionic interaction, van der Waals interaction, or hydrophobicinteraction or a combination thereof.

As used herein, the term “competitive binding” is intended to meanbinding of a first ligand to a binding site of a macromolecule in amanner that prevents a second ligand from binding to the binding site.Accordingly, a first and second ligand that bind to a binding site of amacromolecule in a mutually exclusive manner are understood to becompetitive inhibitors of each other for the macromolecule.

As used herein, the term “bound complex” is intended to mean a specificnon-covalent association between 2 or more molecules. The term caninclude a reversible association so long as the association issufficiently stable to be observed by a binding assay.

As used herein, the term “common ligand” or “CL” is intended to mean amolecule that specifically binds at a site conserved in a family of 2 ormore macromolecules. The term can therefore extend to molecules thatbind to members of a protein family or gene family. Examples of commonligands include a natural common ligand which is normally found inbiological systems or a common ligand mimic which has sufficientstructural similarity to a natural common ligand that it cancompetitively inhibit binding of the natural common ligand to its commonligand binding site. Accordingly, a “common ligand site” is intended tomean a location in or on a macromolecule where a common ligand binds. Acommon ligand site is also referred to as a conserved site.

The term “mimic,” when used in reference to a ligand, is intended tomean a molecule that binds to a protein at the same-site as the ligand.The term can encompass molecules having portions similar tocorresponding portions of the ligand in terms of structure or function.The term can also encompass the original ligand itself.

An example of a useful CL is a cofactor or a cofactor mimic. A“cofactor” is any small molecule that binds in the CL site andparticipates in catalysis when bound to an enzyme. Cofactors oftencontain a nucleotide such as adenine mononucleotide or nicotinamidemononucleotide. Examples of such cofactors include ATP, ADP and SAM(S-adenosyl methionine).

Another group of cofactors that contain a nucleotide is the group NAD⁺,NADH, NADP⁺ and NADPH. Other such cofactors include FMNH₂, FMN, FAD,FADH₂, CoA, GTP and GDP. Still other cofactors include THF, DHF, TPP,biotin, dihydropterin, heme, pyridoxal phosphate and thiaminepyrophosphate. Other common ligands include conserved ligands such asfarnesyl, farnesyl-pyrophosphate, geranyl, geranyl-pyrophosphate orubiquitin.

As used herein, the term “macromolecular target” is intended to mean amacromolecule to which a ligand-probe specifically binds. Amacromolecular target can be, for example, a polypeptide; a nucleic acidmolecule or nucleic acid molecule complex; a polysaccharide; a lipid; ora combination thereof.

As used herein, the term “family,” when used in reference to amacromolecule, is intended to mean a group of at least 2 macromoleculesexhibiting structure homology and at least one function in common. Anexemplary function included in the term is the ability to bind a commonligand such as NADH or ATP. Examples of enzyme families include kinases,dehydrogenases, oxidoreductases, GTPases, carboxyl transferases, acyltransferases, decarboxylases, transaminases, racemases, methyltransferases, formyl transferases, and a-ketodecarboxylases. As usedherein, the term “enzyme” refers to a molecule that binds a substrateligand and carries out a catalytic reaction by converting the substrateligand to a product.

Enzymes can also be classified based on Enzyme Commission (EC)nomenclature recommended by the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (IUBMB) andavailable from the ENZYME database. (available on the internet atexpasy.ch/enzyme/; administered by The Swiss Institute forBioinformatics, Switzerland; see, for example, Bairoch, Nucl. Acid. Res.28:304-305 (2000)). For example, oxidoreductases are classified asoxidoreductases acting on the CH—OH group of donors with NAD⁺ or NADP⁺as an acceptor (EC 1.1.1); oxidoreductases acting on the aldehyde or oxogroup of donors with NAD⁺ or NADP⁺ as an acceptor (EC 1.2.1);oxidoreductases acting on the CH—CH group of donors with NAD or NADP⁺ asan acceptor (EC 1.3.1); oxidoreductases acting on the CH—NH₂ group ofdonors with NAD⁺ or NADP⁺ as an acceptor (EC 1.4.1); oxidoreductasesacting on the CH—NH group of donors with NAD or NADP⁺ as an acceptor (EC1.5.1); oxidoreductases acting on NADH or NADPH (EC 1.6); andoxidoreductases acting on NADH or NADPH with NAD⁺ or NADP⁺ as anacceptor (EC 1.6.1).

Additional oxidoreductases include oxidoreductases acting on a sulfurgroup of donors with NAD⁺ or NADP⁺ as an acceptor (EC 1.8.1);oxidoreductases acting on diphenols and related substances as donorswith NAD⁺ or NADP⁺ as an acceptor (EC 1.10.1); oxidoreductases acting onhydrogen as donor with NAD⁺ or NADP⁺ as an acceptor (EC 1.12.1);oxidoreductases acting on paired donors with incorporation of molecularoxygen with NADH or NADPH as one donor and incorporation of two atoms(EC 1.14.12) and with NADH or NADPH as one donor and incorporation ofone atom (EC 1.14.13); oxidoreductases oxidizing metal ions with NAD⁺ orNADP⁺ as an acceptor (EC 1.16.1); oxidoreductases acting on —CH₂ groupswith NAD or NADP⁺ as an acceptor (EC 1.17.1); and oxidoreductases actingon reduced ferredoxin as donor, with NAD⁺ or NADP⁺ as an acceptor (EC1.18.1).

Other enzymes include transferases classified as transferasestransferring one-carbon groups (EC 2.1);

-   methyltransferases (EC 2.1.1); hydroxymethyl-, formyl- and related    transferases (EC 2.1.2); carboxyl- and carbamoyltransferases (EC    2.1.3); acyltransferases (EC 2.3); and transaminases (EC 2.6.1).    Additional enzymes include phosphotransferases such as    phosphotransferases transferring phosphorous-containing groups with    an alcohol as an acceptor (kinases) (EC 2.7.1); phosphotransferases    with a carboxyl group as an acceptor (EC 2.7.2); phosphotransfer    with a nitrogenous group as an acceptor (EC 2.7.3);    phosphotransferases with a phosphate group as an acceptor (EC    2.7.4); and diphosphotransferases (EC 2.7.6).

Protein or gene family members can often be identified by the presenceof a conserved structural motif as described, for example, in Brandenand Tooze Introduction to Protein Structure, Garland Publishing Inc.,New York (1991). A structural motif can be identified at the primarystructure level according to a particular nucleotide or amino acidsequence or at the tertiary structure level due to a particularcombination or orientation of secondary structure elements.Identification of structural motifs using structural alignments isdescribed in further detail below.

Several large protein and gene families have been identified, includingfamilies having as many as 20 or more, 50 or more, 100 or more and even200 or more members. Two particular examples of a protein or gene familyare kinases and oxidoreductases. The term “kinase” herein means anyenzyme that catalyzes the transfer of a phosphoryl group from ATP orother nucleoside triphosphate to another compound. The term“oxidoreductase” herein means any enzyme that catalyzes anoxidation-reduction reaction. Still other gene families includetransaminases, decarboxylases and methyltransferases.

Another particular gene family is the dehydrogenase gene family. Theterm “dehydrogenase” herein means any enzyme that catalyzes the removalof hydrogen from a substrate using a compound other than molecularoxygen as an acceptor. Typically the hydrogen is transferred to thecoenzyme NAD⁺ (nicotinamide adenine dinucleotide) or NADP⁺ (nicotinamideadenine dinucleotide phosphate). The dehydrogenase gene family is large,containing approximately 17% of all enzymes (You, Kwan-sa,“Stereospecificity for Nicotinamide Nucleotides in Enzymatic andChemical Hydride Transfer Reactions,” CRC Crit. Rev. Biochem. 17:313-451(1985)). Thus, the dehydrogenase family is likely to be a rich source ofdrug targets.

As used herein, the term “specificity” refers to the ability of a ligandto selectively bind to one macromolecule over another. For example, theterm can include selective binding of a ligand to one member of aprotein family compared to other proteins outside of or within theprotein family. The selective binding of a particular ligand to amacromolecule is measurably higher than the binding of the ligand to atleast one other molecule. Specificity can also be exhibited over two ormore, three or more, four or more, five or more, six or more, seven ormore, ten or more, or even twenty or more other macromolecules.

As used herein, the term “structure model” is intended to mean arepresentation of the relative locations of atoms of a molecule. Arepresentation included in the term can be defined by a coordinatesystem that is preferably in 3 dimensions, however, manipulation orcomputation of a model can be performed in 2 dimensions or even 4 ormore dimensions in cases where such methods are desired. The location ofatoms in a molecule can be described, for example, according to bondangles, bond distances, relative locations of electron density, probableoccupancy of atoms at points in space relative to each other, probableoccupancy of electrons at points in space relative to each other orcombinations thereof. A representation included in the term can containinformation for all atoms of a particular molecule or a subset of atomsthereof. Examples of representations included in the term that contain asubset of atoms are those commonly used for polypeptide structures suchas ribbon diagrams, and the like, which show the coordinates of thepolypeptide backbone while omitting coordinates for all or a portion ofthe side chain moieties of the polypeptide. Representations for othermacromolecules and small molecules included in the term can similarlycontain all or a subset of atoms.

A structure model can include a representation that is determined fromempirical data derived from, for example, X-ray crystallography ornuclear magnetic resonance spectroscopy. A representation included inthe term can include one that is derived from a theoretical calculationincluding, for example, a structure obtained by homology modeling or abinitio modeling. A representation of a structure model can include, forexample, an electron density map, atomic coordinates, x-ray structuremodel, ball and stick model, density map, space filling model, surfacemap, Connolly surface, Van der Waals surface or CPK model.

As used herein, the term “docking” is intended to mean using a model ofa first and second molecule to simulate association of the first andsecond molecule at a proximity sufficient for at least one atom of thefirst molecule to be within bonding distance of at least one atom of thesecond molecule. The term is intended to be consistent with its use inthe art pertaining to molecular modeling. A model included in the termcan be any of a variety of known representations of a moleculeincluding, for example, a graphical representation of itsthree-dimensional structure, a set of coordinates, set of distanceconstraints, set of bond angle constraints or set of other physical orchemical properties or combinations thereof.

As used herein, the term “magnetization transfer” is intended to mean athrough-space alteration of the nuclear magnetic resonance properties ofan atomic nucleus of a first atom due to a proximal atomic nucleus or atleast one electron of a proximal atom. An alteration included in theterm can occur due to the Nuclear Overhauser Effect (NOE) or crosssaturation. Proximal atomic nuclei included in the term are those thatare within a distance sufficient to cause a magnetic interactiondetectable by a nuclear magnetic resonance spectroscopy measurement usedin the methods of the invention. Examples of magnetic effects includedin the term are a relaxation effect which can be detected for atoms thatare about 10 Å apart or closer, the Nuclear Overhauser Effect which canbe detected for atoms that are about 6 Å apart or closer or chemicalshift due to shielding or de-shielding which can be detected for atomsthat are about 10 Å or closer. Atoms that are about 5 Å apart or closer,4 Å apart or closer, 3 Å apart or closer, 2 Å apart or closer or 1 Åapart or closer are also proximal atoms that are included in the term.

As used herein, the term “linker” is intended to mean one or more atomsthat covalently connect a first moiety to a second moiety. A moietyincluded in the term can be a ligand such as a common ligand, orfragment thereof; a specificity ligand, or fragment thereof; or a mimicof a common ligand or specificity ligand. A linker can providepositioning and orientation of a first moiety relative to a secondmoiety such that one moiety can bind to a first ligand site and theother moiety can bind to a second, proximal site on a macromolecule.

As used herein, a “library” is intended to mean a population ofdifferent molecules. The library is chemically synthesized and containsprimarily the components generated during the synthesis. A populationincluded in the term can include two or more different molecules. Apopulation can be as large as the number of individual moleculescurrently available to the user or able to be made by one skilled in theart. A population can be as small as two molecules and as large as 10¹⁰molecules. Generally, a population will contain two or more, three ormore, five or more, nine or more, ten or more, twelve or more, fifteenor more, or twenty or more different molecules. A population can alsocontain tens or hundreds of different molecules or even thousands ofdifferent molecules. For example, a population can contain about 20 toabout 100,000 different molecules or more, for example about 25 or more,30 or more, 40 or more, 50 or more, 75 or more, 100 or more, 150 ormore, 200 or more, 300 or more, 500 or more, or 1000 or more differentmolecules, and particularly about 10,000, 100,000 or even 1×10⁶ or moredifferent molecules. A population of synthetic compounds can be derived,for example, by chemical synthesis and is substantially free ofnaturally occurring substances.

As used herein, the term “homolog” is intended to mean a molecule ormoiety of a molecule that has similar structure in comparison to areference molecule or moiety. A moiety is a group of atoms that form apart or portion of a larger molecule. A moiety can consist of any numberof atoms in a portion of a molecule and can correlate with a physical orchemical property conferred upon the molecule by the combined atoms.

As used herein, the term “ligand-probe” is intended to mean a moleculethat can selectively bind a protein and that has an antenna moiety and aligand moiety. A “ligand moiety” is a fragment of a ligand-probe, thatwhen lacking the antenna moiety, is capable of selectively binding tothe protein. An “antenna moiety” is a structure containing anNMR-visible nucleus that is attached to a ligand moiety by bonding to atleast 1 intervening atoms. A larger number of atoms can intervenebetween an NMR-visible nucleus and ligand moiety including, for example,at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more intervening atoms. Theintervening atoms can form an aliphatic chain that, when attached to aligand moiety having aromatic rings, allows selective excitation due todifferences in frequency for excitation or saturation of aliphatic andaromatic protons. An NMR-visible nucleus of an antenna moiety can be aproton that is isolated from vicinal proton coupling. Isolation fromvicinal proton coupling provides for selective observation of direct NOEtransfer at short mixing times compared to indirect NOE transfer viaspin diffusion and also reduces signal loss due to relaxation effectsthat occur for vicinal coupled protons. A proton can be isolated fromvicinal proton coupling by being attached to a carbon that is adjacentto an atom that lacks protons including, for example, an ether oxygen;carbonyl carbon; thioether, sulfone or sulfoxide sulfur; deuteratedcarbon or selenium, or by being attached to a carbon that is adjacent toan atom having fast exchanging protons such as a nitrogen at high pH. AnNMR-visible nucleus of an antenna moiety also can be, for example, ¹³C,¹⁵N, ¹⁹F, ³¹P, or ¹¹³Cd. Such a nucleus can be isolated for selectiveobservation of direct NOE transfer using a variety of isotope-editingNMR methods. As shown in example VII, ¹³C-isotope editing was used toisolate coupling of a carbon in an antenna moiety of a ligand-probe to aportion of a ligand. A probe ligand or antenna moiety can contain two ormore NMR-visible nuclei, such as two or more ¹³C, ¹⁵N, ¹⁹F, ³¹P, or¹¹³Cd-enriched heteroatoms. The presence of such heteroatoms can beuseful for the application of methods for obtaining 3D X-nucleusseparated NOESY spectra, such as 3D HMQC/HSQC-NOESY or NOESY-HMQC/HSQCspectra to separate IL-NOE peaks corresponding to each isotope enrichedand attached proton into different planes in the spectrum. Such methodscan be used to resolve signal over-lap in the two proton dimensions. Foruse in isotope edited methods, the choice of where to incorporate aparticular nuclei into a ligand-probe or antenna moiety and the choiceof the number of enriched heteroatoms to include, can be determined bythose skilled in the art based on physical properties of the particularligand-probes or antenna moieties.

The invention provides a method for assembling a binding compound. Themethod includes the steps of (a) obtaining a sample containing amacromolecule, a first ligand and a second ligand under conditionswherein a bound complex is formed containing the first ligand, thesecond ligand and the macromolecule; (b) providing a sample comprisingthe protein, the first ligand and a second ligand under conditionswherein the first ligand, the second ligand and the protein form a boundComplex; (c) detecting a subset of magnetization transfer signalsbetween the first ligand and the second ligand in the bound complex,wherein the signals are obtained from an isotope edited NOESY spectrumof the sample; thereby determining that the two ligands are proximal inthe bound complex; and (d) obtaining a population of candidate bindingcompounds comprising the first ligand, or a fragment thereof, linked toone of a plurality of second ligand homologs, whereby the populationcontains binding compounds that bind to members of the protein family.

A schematic overview that exemplifies a method for assembling a bindingcompound is shown in FIG. 1. At step 1, a ligand, shown as F₁, isidentified based on the observation that it binds to a protein. Thebinding can be observed based on magnetization transfer between theprotein binding site and the F_(i) ligand. The F₁ ligand can be obtainedfrom a screen in which a library of candidate ligands are tested for theability to bind the protein. At step 2, the F₂ ligand is identified asbinding to the protein at a location that is proximal to the F₁ ligand.Proximal ligands can be identified based on the observation ofmagnetization transfer between the F₁ ligand and F₂ ligand when in acomplex with the protein. The F₂ ligand can be identified from a screenusing a library of candidate ligands. Based on the observedmagnetization transfer a bi-ligand compound can be obtained in which theF₁ and F₂ ligands are linked. Alternatively, fragments or homologs ofeither ligand can be linked to form a bi-ligand. As shown in step 2′,the bi-ligand compound can be used to identify a third proximal ligand,shown as ligand F₃, and a tri-ligand can be subsequently obtained inwhich the F₁, F₂ and F₃ ligands are linked. Similarly, an F₁ ligand canbe identified in step 1, while an F₂ ligand is identified in step 2.

A schematic overview that exemplifies another method for assembling abinding compound is shown in FIG. 21. FIG. 21 shows a previouslyidentified binding compound (A) and subsequent separation of the bindingcompound into F₁ and F₂ fragments (B). In FIG. 21C, the F₂ fragment isused to identify a different proximal F1 ligand. In FIG. 21D, the F₂fragment is used to identify an F₂′ proximal ligand that binds to adifferent binding site. The distance between a newly identified F₂′proximal ligand and F₂ or F₁ can be determined if the binding sites areclose enough to allow inter-ligand NOEs to be observed between them, andbased on knowledge of the position of the original F₁. Thus, theposition of one or more new F₂′ fragments from the inter-ligand NOEsbetween the an F₂ fragment and a new F₂′ binding site remote from theoriginal F₁ can be determined by “triangulation.” As described herein, avariety of strategies can be used for identifying distances betweenligand fragments, which can include “Forward NMR ACE,” used when acommon ligand is used to probe for another ligand, such as a specificityligand; “Reverse NMR ACE,” used when a specificity ligand is used toprobe for a proximal ligand, such as a common ligand; “Triangulation,”used to identify a third ligand (F₂′ fragment) from two known F1fragments, two known F₂ fragments, or one of each; and “Extended NMRACE,” used to identify a new F₂′ that binds remote from the F₁ from anF₂ ligand-probe. These and related methods are set forth in furtherdetail below.

Initially, a macromolecule target such as a protein is identified forthe development of a binding compound. In one embodiment, amacromolecule target for development of a therapeutic agent can beidentified based on its presence in a pathogen or its association with adisease or condition. For example, a protein target present in apathogen can be selected as the target to develop drugs effective incombating a disease caused by that pathogen. Any pathogen can beselected as a target organism. Examples of pathogens include, forexample, bacteria, fungi or protozoa.

Pathogenic bacteria useful as target organisms include Staphylococcus,Mycobacteria, Mycoplasma, Streptococcus, Haemophilus, Neisseria,Bacillus, Clostridium, Corynebacteria, Salmonella, Shigella, Vibrio,Campylobacter, Helicobacter, Pseudomonas, Legionella, Bordetella,Bacteriodes, Fusobacterium, Yersinia, Actinomyces, Brucella, Borrelia,Rickettsia, Ehrlichia, Coxiella, Chlamydia, and Treponema. Pathogenicstrains of Escherichia coli can also be target organisms.

Binding compounds targeted to macromolecules in these pathogenicbacteria are useful for treating a variety of diseases includingbacteremia, sepsis, nosocomial infections, pneumonia, pharyngitis,scarlet fever, necrotizing fasciitis, abscesses, cellulitis, rheumaticfever, endocarditis, toxic shock syndrome, osteomyelitis, tuberculosis,leprosy, meningitis, pertussis, food poisoning, enteritis,enterocolitis, diarrhea, gastroenteritis, shigellosis, dysentery,botulism; tetanus, anthrax, diphtheria, typhoid fever, cholera,actinomycosis, Legionnaire's disease, gangrene, brucellosis, Lymedisease, typhus, spotted fever, Q fever, urethritis, vaginitis,gonorrhea and syphilis.

For example, Staphylococcus aureus is a major cause of nosocomialinfections and has become increasingly resistant to a variety ofantibiotics over recent years. Similarly, Mycobacteria tuberculosis hasbecome increasingly resistant to multiple antibiotics in recent years.M. tuberculosis infects almost one third of the world population, withactive tuberculosis found in almost 10 million people worldwide and inAIDS patients as a common opportunistic infection. Streptomyces has alsobecome increasingly resistant to antibiotics over recent years.Therefore, these pathogenic bacteria with known resistance and targetmacromolecules required for their growth or pathogenesis areparticularly desirable as target organisms for which therapeutic bindingcompounds can be identified.

In another embodiment, target organisms are selected from yeast andfungi. Pathogenic yeast and fungi useful as target organisms includeAspergillus, Mucor, Rhizopus, Candida, Cryptococcus, Blastomyces,Coccidioides, Histoplasma, Paracoccidioides, Sporothrix, andPneumocystis. Binding compounds targeted to macromolecules in thesepathogenic yeast and fungi are useful for treating a variety of diseasesincluding aspergillosis, zygomycosis, candidiasis, cryptococcoses,blastomycosis, coccidioidomycosis, histoplasmosis,paracoccidioidomycosis, sporotrichosis, and pneuomocystis pneumonia.

In still another embodiment, target organisms are selected fromprotozoa. Pathogenic protozoa useful as target organisms includePlasmodium, Trypanosoma, Leishmania, Toxoplasma, Cryptosporidium,Giardia, and Entamoeba. Binding compounds targeted to macromolecules inthese pathogenic protozoa are useful for treating a variety of diseasesincluding malaria, sleeping sickness, Chagas' disease, leishmaniasis,toxoplasmosis, cryptosporidiosis, giardiasis, and amebiasis.

In addition, a target cell such as a cancer cell can be selected toidentify drugs effective for treating cancer. Examples of such targetcells include, for example, breast cancer, prostate cancer, and ovariancancer cells as well as leukemia, lymphomas, melanomas, sarcomas andgliomas. Binding compounds directed to a target macromolecule in acancer cell are useful for targeted delivering of a chemotherapeuticagent or for inhibition of unregulated growth. Diagnosis andidentification of causative factors or pathogens for a targeted diseasecan be determined using methods known in the art as described forexample in The Merck Manual, Sixteenth Ed, (Berkow, R., Editor) Rahway,N.J., 1992.

A macromolecule family to which a target macromolecule belongs can beidentified according to structural or functional similarities usingmethods known in the art. Structural similarity can be identified, forexample, by sequence analysis at the nucleotide or amino acid level. Onemethod for determining if two macromolecules are related is BLAST, BasicLocal Alignment Search Tool. (available on the internet atncbi.nlm.nih.gov/BLAST/; administered by The National Center, forBiotechnology Information, Bethesda Md.). BLAST is a set of similaritysearch programs designed to examine all available sequence databases andcan function to search for similarities in protein or nucleotidesequences. A BLAST search provides search scores that have awell-defined statistical interpretation. Furthermore, BLAST uses aheuristic algorithm that seeks local alignments and is therefore able todetect relationships among sequences which share only isolated regionsof similarity (Altschul et al., J. Mol. Biol. 215:403-410 (1990)).

In addition to the originally described BLAST (Altschul et al., supra,1990), modifications to the algorithm have been made (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)). One modification is GappedBLAST, which allows gaps, either insertions or deletions, to beintroduced into alignments. Allowing gaps in alignments tends to reflectbiologic relationships more closely. A second modification is PSI-BLAST,which is a sensitive way to search for sequence homologs. PSI-BLASTperforms an initial Gapped BLAST search and uses information from anysignificant alignments to construct a position-specific score matrix,which replaces the query sequence for the next round of databasesearching. A PSI-BLAST search is often more sensitive to weak butbiologically relevant sequence similarities.

A second resource for identifying members of a protein family isPROSITE. (Available on the internet at expasy.ch/sprot/prosite.html;administered by The Swiss Institute for Bioinformatics, Switzerland).PROSITE is a method of determining the function of uncharacterizedproteins translated from genomic or cDNA sequences (Bairoch et al.,Nucleic Acids Res. 25:217-221 (1997)). PROSITE consists of a database ofbiologically significant sites and patterns that can be used to identifywhich known family of proteins, if any, the new sequence belongs. Insome cases, the sequence of an unknown protein is too distantly relatedto any protein of known structure to detect its resemblance by overallsequence alignment. However, related proteins can be identified by theoccurrence in its sequence of a particular cluster of amino acidresidues, which can be called a pattern, motif, signature orfingerprint. PROSITE uses a computer algorithm to search for motifs thatidentify proteins as family members. PROSITE also maintains acompilation of previously identified motifs, which can be used todetermine if a newly identified protein is a member of a known proteinfamily.

Members of a protein family can also be identified by clustering bindingsite structures or bound ligand conformations as described, for example,in U.S. patent application Ser. No. 10/040,895. A sequence model such asa Hidden Markov Model, representing the frequency and order with whichspecific amino acids or gaps occur in the binding sites of proteinfamily members can be used to search a sequence database and identifyother members as described, for example, in U.S. patent application Ser.No. 10/040,895. Members of a protein family can also be identified byclustering their sequence comparison signatures, where a sequencecomparison signature for a protein is a string of pairwise comparisonscores for the protein compared to the other proteins in a database asdescribed, for example, in U.S. patent application Ser. No. 10/032,395.

Another resource for identifying members of a protein family isStructural Classification of Proteins (SCOP, Available on the internetat scop.mrc-lmb.cam.ac.uk/scop/, administered by Medical Researchcouncil, Cambridge, England. (which is incorporated herein byreference). SCOP maintains a compilation of previously determinedprotein tertiary folds from which structural comparison can be made toidentify protein family members having similar motifs (Murzin et al., J.Mol. Biol. 247:536-540 (1995)).

TABLE 1 Databases for Identifying Protein Family Motifs SEARCHABLE MOTIFAND PATTERN DATABASES WEBSITES PROSITEexpasy.hcuge.ch/sprot/prosite.html BLOCKSblocks.fhcrc.org/blocks_search.html PRINTSbiochem.ucl.ac.uk/bsm/dbbrowser/PRINTS/PRINTS.html PIMAdot.imgen.bcm.tmc.edu:9331/seq-search/protein- search.html PRODOMprotein.toulouse.inra.fr/prodom.html REGULAR EXPRESSION SEARCHibc.wustl.edu/fpat/ PROFILESEARCH segnet.dl.ac.uk/hhg/PROFILESE.htmlPATSCAN c.mcs.anl.gov/home/overbeek/PatScan/HTML/patscan.html PATTERNFIND ulrec3.unil.ch/software/PATFND-mailform.html PROFILElenti.med.umn.edu/MolBio_man/chp-10.html#HDR1 PMOTIFalces.med.umn.edu/pmotif.html HMMER genome.wustl.edu/eddy/HMMER/ WWW ANDFTP SERVERS FOR SINGLE SEQUENCE EXHAUSTIVE DATABASE SEARCHES WEBSITESBLAST ncbi.nlm.nih.gov/BLAST/ BLITZ ebi.ac.uk/searches/blitz_input.htmlFASTA genome.ad.jp/ideas/fasta/fasta_genes.html FTP ADDRESSES FOR MOTIFAND PROFILE SEARCH PROGRAMS WESITES BARTON'S FLEXIBLE PATTERNSgeoff.biop.ox.ac.uk/ PROPAT mdc-berlin.de/ SOMftp.mdc-berlin.de/pub/neural SEARCHWISE sable.ox.ac.uk/pub/users PROFILEftp.ebi.ac.uk/pub/software/unix/ TPROFILESEARCHftp.ebi.ac.uk/pub/softare/vax/egcg CAP nin/capncbi.nlm.nih.gov/pub/koonin/cap

Additional resources for identifying motifs of a protein family areshown in Table 1. The websites cited therein are incorporated byreference.

Conserved amino acids are evolutionarily conserved and carry out acommon function. For example, the Rossman fold is a tertiary structuralmotif that includes GXXGXXG or GXGXXG and is present in enzymes thatbind nucleotides (Brandon and Tooze, in Introduction to ProteinStructure, Garland Publishing, New York (1991)). Enzymes that bindnucleotides such as NAD, NADP, FAD, ATP, ADP, AMP and FMN contain theRossman fold sequence motif (Creighton, Proteins: Structures andMolecular Principles, p. 368, W.H. Freeman, New York (1984)). Additionalconserved residues as well as different protein structures distinguishprotein families that bind, for example, NAD from those that bind, forexample, ATP.

An example of a recognizable protein motif or fingerprint is found indinucleotide binding proteins such as dehydrogenases (Rossman et al., inThe Enzymes Vol 11, Part A, 3rd ed., Boyer, ed., pp. 61-102, AcademicPress, New York (1975); Wierenga et al., J. Mol. Biol. 187:101-107(1986); and Ballamacina, FASEB J. 10:1257-1269 (1996)). The fingerprintregion contains a phosphate binding consensus sequence GXXGXXG orGXGXXG, a hydrophobic core of six small hydrophobic residues, aconserved, negatively charged residue that binds to the ribose 2′hydroxyl of adenine and a conserved positively charged residue(Bellamacina, supra).

Protein kinases also have recognizable motifs conserved among all knownprotein kinases (Hanks and Quinn, Methods Enzymol. 200:28-62 (1991)).Eight invariant amino acid residues are conserved throughout the proteinkinase family, including a conserved GXGXXG motif similar to that seenin dinucleotide binding proteins. A crystallographic molecular model ofcyclic AMP-dependent protein kinase as well as other protein kinasesshowed that these conserved residues are nearly all associated withessential, conserved functions such as ATP binding and catalysis(Knighton et al., Science 253:407-414 (1991); and Knighton et al.,Science 253:414-420 (1991)). Thus, conserved amino acid residues, whichare common to members of a protein family, are recognizable as a motifcritical for the structure, function or activity of a protein.

-   -   Pyridoxal binding proteins also have recognizable motifs. One        motif is GXGGXXXG (SEQ ID NO: 2), a second motif is        KXEX₆SXKX₅₋₆M (SEQ ID NO: 3), and a third motif is PXNPTG (SEQ        ID NO: 4) (Suyama et al., Protein Engineering 8:1075-1080        (1995)).

A macromolecule family can be selected based on a conserved andrecognizable structural motif such as a primary sequence motif, tertiarystructure motif, or both. Members of a macromolecule family can also berecognized based on similar function. For example, a protein family canbe identified based on the ability of its members to bind a naturalcommon ligand that is already known. For example, it is known thatdehydrogenases bind to dinucleotides such as NAD or NADP. Therefore, NADor NADP are natural common ligands to a number of dehydrogenase familymembers. Similarly, kinases bind ATP, which is therefore a naturalcommon ligand to kinases. Other natural common ligands of amacromolecule family can be the coenzymes and cofactors described above.

After a target macromolecule is selected, the selected macromolecule ora functional fragment thereof cam be isolated for use in the methods. Afunctional fragment of a macromolecule is a fragment that is capable ofbinding at least one ligand that is bound by the full lengthmacromolecule. The macromolecule or fragment can be isolated from anative tissue or organism, from a population of cells maintained inculture, or from a recombinant organism or cell culture. Methods forisolating a protein are known in the art and are described, for example,in Scopes, Protein Purification: Principles and Practice, 3^(rd) Ed.,Springer-Verlag, New York (1994); Duetscher, Methods in Enzymology, Vol182, Academic Press, San Diego (1990); and Coligan et al., Currentprotocols in Protein Science, John Wiley and Sons, Baltimore, Md.(2000).

A target macromolecule can be cloned and expressed in a recombinantorganism using methods that are known to those skilled in the artincluding, for example, polymerase chain reaction (PCR) and othermolecular biology techniques (Dieffenbach and Dveksler, eds., PCRPrimer: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Plainview, N.Y. (1995); Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.(1989); Ausubel et al., Current Protocols in Molecular Biology, Vols.1-3, John Wiley & Sons (1998)). The gene or cDNA encoding the targetmacromolecule is cloned into an appropriate expression vector forexpression in an organism such as bacteria, insect cells, yeast ormammalian cells.

Appropriate expression vectors include those that are replicable ineukaryotic cells and/or prokaryotic cells and can remain episomal or beintegrated into the host cell genome. Suitable vectors for expression inprokaryotic or eukaryotic cells are well known to those skilled in theart as described, for example, in Ausubel et al., supra. Vectors usefulfor expression in eukaryotic cells can include, for example, regulatoryelements including the SV40 early promoter, the cytomegalovirus (CMV)promoter, the mouse mammary tumor virus (MMTV) steroid-induciblepromoter, Moloney murine leukemia virus (MMLV) promoter, and the like. Avector useful in the methods of the invention can include, for example,viral vectors such as a bacteriophage, a baculovirus or a retrovirus;cosmids or plasmids; and, particularly for cloning large nucleic acidmolecules, bacterial artificial chromosome vectors (BACs) and yeastartificial chromosome vectors (YACs). Such vectors are commerciallyavailable, and their uses are known in the art. One skilled in the artwill know or can readily determine an appropriate promoter forexpression in a particular host cell.

If desired, a target protein can be expressed as a fusion with anaffinity tag that facilitates purification of the target protein. Forexample, the target protein can be expressed as a fusion with a poly-Histag, which can be purified by metal chelate chromatography. Other usefulaffinity purification tags which can be expressed as fusions with thetarget protein and used to affinity purify the protein include, forexample, a biotin, polyhistidine tag (Qiagen; Chatsworth, Calif.),antibody epitope such as the flag peptide (Sigma; St Louis, Mo.),glutathione-S-transferase (Amersham Pharmacia; Piscataway, N.J.),cellulose binding domain (Novagen; Madison, Wis.), calmodulin(Stratagene; San Diego, Calif.), staphylococcus protein A (Pharmacia;Uppsala, Sweden), maltose binding protein (New England BioLabs;Beverley, Mass.) or strep-tag (Genosys; Woodlands, Tex.) or minormodifications thereof.

A target macromolecule can be validated as a representative member of amacromolecule family. In some cases, the target macromolecule is wellcharacterized with respect to its binding properties to a natural commonligand. However, if the target macromolecule is encoded by a new,relatively uncharacterized gene, the expressed target macromolecule canbe tested to confirm that it binds the natural common ligand. Othercommon ligands of related macromolecule families, for example, othernucleotide binding macromolecules, or known ligand mimics can also betested for binding to the target macromolecule.

A target macromolecule can be further validated as a useful therapeutictarget by determining if the selected target macromolecule is known tobe required for normal growth, viability or infectivity of the targetorganism or cell. If it is unknown whether the target macromolecule isrequired for normal growth, viability, or infectivity, the targetmacromolecule can be specifically inactivated by gene knockout, in amodel organism to determine if the macromolecule performs a criticalfunction required for survival or infectivity of the organism or cell.Such a macromolecule providing a critical function is a good target fordeveloping therapeutic agents.

Methods for disrupting a gene to generate a knockout are well known inthe art (Ausubel et al., Current Protocols in Molecular Biology, Vols1-3, John Wiley & Sons (1998)). For example, transposable elements canbe used to knockout a gene and test for the effect of the knockout oncell growth, viability or infectivity (Benson and Goldman, J. Bacteriol.174:1673-1681 (1992); Hughes and Roth, Genetics 119:9-12 (1988); andElliot and Roth, Mol. Gen. Genet. 213:332-338 (1988)). Methods for geneknockouts in protozoa have also been previously described (Wang,Parasitology 114:531-544 (1997); and Li et al, Mol. Biochem. Parasitol.78:227-236 (1996)).

Although use of the methods of the invention is exemplified herein withregard to proteins, it is understood that a method of the invention canbe used for any other macromolecule that is capable of binding two ormore ligands in proximity. Other macromolecules include, for example,biological polymers such as polysaccharides or polynucleotides orsynthetic polymers such as plastics and mimics of biological polymers. Apolynucleotide can be, for example, a ribozyme, ribosomal RNA or otherRNA that is capable of binding a ligand such as a nucleotide.

A method of the invention can include a step of identifying a commonligand. In some cases, a common ligand to a macromolecule family isalready known. For example, NAD is a natural common ligand fordehydrogenases, and ATP is a natural common ligand for kinases. However,natural common ligands such as the coenzymes and cofactors often havelimitations regarding their usefulness as a starting compound.Substrates and cofactors often undergo a chemical reaction, for example,transfer of a group to another substrate or reduction or oxidationduring the enzymatic reaction. However, it is desirable that a ligand tobe used as a drug is not metabolizable. Therefore, a natural commonligand or a derivative thereof that is non-metabolizable is generallypreferred as a common ligand. Examples of mimetics to the common ligandNADH, for example cibacron blue, are described in Dye-LigandChromatography, Amicon Corp., Lexington Mass. (1980). Numerous otherexamples of NADH-mimics, including useful modifications to obtain suchmimics, are described in Everse et al. (eds.), The Pyridine NucleotideCoenzymes, Academic Press, New York N.Y. (1982).

A ligand that binds a macromolecule can be identified or characterizedusing a binding assay including, for example, an equilibrium bindinganalysis, competition assay, or kinetic assay as described in Segel,Enzyme Kinetics John Wiley and Sons, New York (1975), and Kyte,Mechanism in Protein Chemistry Garland Pub. (1995). A common ligand canbe identified by a competitive binding assay. For example, amacromolecule can be incubated in the presence of a known common ligandand a candidate common ligand, and the rate or extent to which thecommon ligand binds the macromolecule can be determined. Competitivebinding between the common ligand and candidate ligand can be identifiedfrom a reduction in the rate or extent of binding of the common ligandto the macromolecule in the presence of the candidate ligand, comparedto in the absence of the candidate ligand (see, for example, Segel,Enzyme Kinetics John Wiley and Sons, New York (1975)). A candidateligand that competes with the known common ligand for binding to thecommon ligand site on the macromolecule is identified as a new commonligand.

Alternatively, absence of competitive binding of a ligand for the siteon a protein to which a common ligand binds can be used to determinethat a ligand binds to a different location on the protein from thecommon ligand such as a site that is external to the common ligandbinding site. A site that is external to the common ligand binding siteon a protein can be, for example, a specificity ligand binding site.Binding at a specificity ligand binding site can be determined bycompetitive binding with a specificity ligand or mimic thereof. Even ifthe location where a ligand binds is not known, a determination that theligand binds to a different location of a protein compared to a secondligand combined with information regarding proximity of the two ligandscan be used to map the protein binding site, as demonstrated in ExampleIII. A ligand, site on a protein that is identified as external to acommon ligand binding site provides a specificity target for which abinding compound can be designed. Such a binding compound designed tointeract with the external site will typically show selective binding tothe protein compared to other proteins that bind the same common ligand.

In some cases, a common ligand has an intrinsic property that is usefulfor detecting whether it is bound. For example, the natural commonligand for dehydrogenases, NAD, has intrinsic fluorescence. Therefore,increased fluorescence in the presence of candidate common ligands dueto displacement of NAD can be used to detect competition for binding ofNAD to a target NAD binding macromolecule (Li and Lin, Eur. J. Biochem.235:180-186 (1996); and Ambroziak and Pietruszko, Biochemistry28:5367-5373 (1989)).

In other cases, when the common ligand does not have an intrinsicproperty useful for detecting ligand binding, it can be labeled with adetectable moiety. For example, the natural common ligand for kinases,ATP, can be radiolabeled with ³²P, and the displacement of radioactiveATP from an ATP binding protein in the presence of a candidate ligandcan be used to identify the candidate as a common ligand. Any detectablemoiety, for example a radioactive or fluorescent label, can be added toa ligand so long as the labeled ligand can bind to its binding site on amacromolecule.

A library of candidate ligands can be screened to identify a ligand thatbinds to a macromolecule, for example, at a common ligand site. Thus, amethod of the invention can include assaying a population of candidatefirst ligands for the ability to bind to a target macromolecule andidentifying from the population of candidate first ligands a firstligand that binds to the macromolecule. A first ligand identified fromsuch a screen can then be used to form a complex with the macromoleculeand a second ligand that binds proximal to the first ligand can beidentified. The screen can be performed by a competitive binding assayon a sample containing the macromolecule, a candidate first ligand and aknown common ligand such that a first ligand can be identified as acommon ligand by its ability to displace the known common ligand.

A library of candidate ligands can contain a broad range of compounds ofvarious structures. However, the library of candidate ligands can alsobe focused on compounds that are more likely to bind to a particularsite in a macromolecule. A focused library can be designed, for example,to have members that are structural homologs of a natural common ligandor that contain moieties found in the common ligand. A library ofcandidate common ligands can also be chosen to include members havingstructural features that are commonly found in a particular class ofligands including, for example, a MOTIF library as described in ExampleII. The library of candidate common ligands can be a group of analogs ormimetics of the natural common ligand.

One approach to identify a common ligand from a library of candidateligands is to perform high throughput screening on a large library ofmolecules. The molecules can be obtained from an existing source such asa commercial or proprietary library or can be synthesized using acombinatorial synthetic method. The iterative approach to combinatorialsynthesis is well-known in the art and is set forth, in general, inHoughten et al., Nature, 354, 84-86 (1991); and Dooley et al., Science,266, 2019-2022 (1994). In the iterative approach, for example,sublibraries of a molecule having three variable groups are made whereinthe first variable is defined. Each of the compounds with the definedvariable group is reacted with all of the other possibilities at theother two variable groups. These sub-libraries are each tested forbinding to the target macromolecule to define the identity of the secondvariable in the sub-library having the highest affinity. A newsub-library with the first two variable positions defined is reactedagain with all the other possibilities at the remaining undefinedvariable position. As before, the identity of the third variableposition in the sub-library having the highest activity is determinedwith a binding assay using the target macromolecule. If more variablesexist, this process is repeated for all variables, yielding the compoundwith each variable contributing to the desired binding affinity in thescreening process. Promising compounds from this process can then besynthesized on larger scale in traditional single-compound syntheticmethods for further biological investigation.

The positional-scanning approach has been described for various organiclibraries and for various peptide libraries (see, for example, R.Houghten et al. PCT/US91/08694 and U.S. Pat. No. 5,556,762). In thepositional scanning approach, sublibraries are made defining only onevariable with each set of sublibraries and all possible sublibrarieswith each single variable defined (and all other possibilities at all ofthe other variable positions) are made and tested. For example, oneskilled in the art could synthesize libraries wherein 2 fixed positionsare defined at a time. From the testing of each single-variable definedlibrary for binding to the target macromolecule, the optimum substituentat that position is determined, pointing to the optimum or at least aseries of compounds having a maximum affinity. Thus, the number ofsublibraries for compounds with a single position defined will be thenumber of different substituents desired at that position, and thenumber of all the compounds in each sublibrary will be the product ofthe number of substituents at each of the other variables.

Once a library of candidate common ligands is selected, the library isscreened, for example, by competition with a natural common ligand forbinding to a target macromolecule, to identify at least one commonligand in the library that binds to a conserved site in the targetmacromolecule. A common ligand identified by the screen is then furthercharacterized with respect to affinity for the target macromolecule. Insome cases it is desirable to identify a common ligand that is not ahigh affinity ligand. Since the common ligand binds to multiple membersof a macromolecule family, a high affinity common ligand would likelybind to other members of the family in addition to the targetmacromolecule. It can therefore be desirable in such cases to identify acommon ligand having modest affinity, preferably at or below theaffinity of the natural common ligand that binds to the same conservedsite. Such a common ligand having modest affinity is then used as astarting compound for identifying a binding compound. Generally, amodest affinity ligand will have affinity for a macromolecule with anequilibrium dissociation constant of about 10⁻² to 10⁻⁷ M, or about 10⁻³to 10⁻⁶ M. The equilibrium dissociation constant of a common ligand orother ligand for a target macromolecule can be greater than 1×10⁻⁶ M.

Another approach to identify a common ligand is to use thethree-dimensional structure of a natural common ligand and search adatabase of commercially available molecules such as the AvailableChemicals Directory (MDL Information Systems, Inc.; San Leandro Calif.)to identify candidate common ligands having similar shape orelectrochemical properties of the natural common ligand. Methods foridentifying similar molecules are well known in the art and arecommercially available (Doucet and Weber, in Computer-Aided MolecularDesign: Theory and Applications, Academic Press, San Diego Calif.(1996); software is available from Molecular Simulations, Inc., SanDiego Calif.). A database can be searched, for example, by queryingbased on chemical property information or on structural information. Inthe latter approach, an algorithm based on finding a match to a templatecan be used as described, for example, in Martin, “Database Searching inDrug Design,” J. Med. Chem. 35:2145-2154 (1992).

Furthermore, if structural information is available for the conservedsite in the macromolecule, particularly with a known ligand bound,compounds that fit the conserved site can be identified throughcomputational methods (Blundell, Nature 384 Supp:23-26 (1996)). Usingsuch an approach a common ligand can be identified by obtaining astructure model for the binding site of the macromolecule and docking astructure model of a candidate ligand with the structure model of thebinding site. Algorithms available in the art for fitting a ligandstructure to a protein binding site include, for example, DOCK (Kuntz etal., J. Mol. Biol. 161:269-288 (1982)) and INSIGHT98 (MolecularSimulations Inc., San Diego, Calif.).

A molecular structure can be conveniently stored in a computer readablemedium and manipulated in a computer system using structuralcoordinates. Structural coordinates can occur in any format known in theart so long as the format can provide an accurate reproduction of theobserved structure. For example, crystal coordinates can occur in avariety of file types such as .fin, .df, .phs, or .pdb as described forexample in McRee et al., Practical Protein Crystallography, AcademicPress, San Diego (1993). Although the examples above describe structuralcoordinates derived from X-ray crystallographic analysis, one skilled inthe art will recognize that structural coordinates can be in any formatderived from or used in a method known in the art for determiningmolecular structure.

A ligand that binds to a target protein can be identified using nuclearmagnetic resonance methods. For example, ligand binding can becharacterized qualitatively or quantitatively by measuringcross-saturation between the ligand and macromolecule when bound in acomplex. An example of a cross-saturation method is WaterLOGSY in whichselective water excitation is followed by NOE mixing such thatmagnetization is effectively transferred via the protein-ligand complexto the free ligand in a selective manner. Under these conditions theresonance of non-bound molecules have an opposite sign and tend to beweaker than the resonances for bound ligands. The macromoleculeresonances can be suppressed with a double spin echo scheme, which alsosuppresses water, and for small and medium sized proteins, where doublespin echo may not sufficiently suppress protein sequences, a T_(1ρ)filter can be introduced into the pulse sequence prior to theacquisition period. Thus, the resonances for bound ligands can bereadily resolved from unbound molecules and the target macromolecule.Accordingly, WaterLOGSY can be used to screen mixtures of potentialligands to identify those that bind to a target macromolecule, forexample, in a screening format. WaterLOGSY is described in furtherdetail in Dalvit et al., J. Biomol. NMR 21:349-359 (2001). Nuclearmagnetic resonance can also be used to identify a ligand that binds amacromolecule by observing changes in line widths, relaxation rates orNOE values for a ligand upon binding to a macromolecule, as described,for example, in Ni et al., Prog. Nucl. Magn. Reson. Spectrosc.26:517-606 (1994).

Two ligands that bind simultaneously to a macromolecule and in closeproximity to each other can be identified in a method of the invention.One of the ligands can be a common ligand, as set forth above. Thesecond ligand can be any molecule that is capable of binding to themacromolecule in proximity with the first ligand at a site that isexternal to the common ligand binding site. In the case of an enzymetarget, a substrate that is acted upon by a cofactor usually provides areasonable candidate as a second ligand. In particular, the commonligand site and substrate site are most likely located in physicalproximity to each other in an enzyme's three-dimensional structure tofacilitate catalysis. In particular, the three-dimensional geometricrelationship between the common ligand site and substrate ligand siteshas been shown to be conserved in evolutionarily related dehydrogenases(Sem and Kasper, Biochemistry 31:3391-3398 (1992)). Although therelationship between the sites is conserved, the substrate site itselfimparts molecular properties that distinguish the protein from otherproteins in the same protein family. Thus, the substrate site isreferred to as a “specificity site.” The specificity site of amacromolecule provides a binding site for a ligand that selectivelyassociates with the macromolecule compared to other macromolecules thatare in the same common ligand-binding family. A site that is external toa common ligand binding site such as a substrate specificity site can beexploited as a potential binding site for the identification of a ligandthat has specificity for one macromolecule over another member of thesame macromolecule family. A site that is external to a common ligandbinding site such as a specificity site is distinct from the commonligand binding site in that the natural common ligand does not bind tothe specificity site.

A second ligand such as a specificity ligand can be identified using theabove-described methods including, for example, a binding assay, astructural characterization or database search. In the case where one orboth ligands are known to bind to a macromolecule, a method of theinvention can be used to determine that the two ligands bind inproximity to each other. Furthermore, as set forth below the relativeorientation of or distance between the two ligands can be determined andused to design a binding compound or a library of candidate bindingcompounds.

A method of the invention can also be used in a screening format toidentify a second ligand that is capable of binding to a macromoleculesimultaneously with a first ligand and in proximity to the first ligand.Thus, a second ligand that has not been previously shown to bind to themacromolecule can be identified as being capable of binding themacromolecule based on detection of an interaction with another ligand.A second ligand can be identified from a library of candidate ligands ina screening method. A second ligand can be any type of ligand inproximity to a first ligand, including a common ligand or specificityligand.

Accordingly, the invention provides a method for obtaining a focusedlibrary of candidate binding compounds, wherein the members of theprotein family bind a common ligand. The method includes the steps of amethod for obtaining a focused library of candidate binding compounds,wherein the members of the protein family bind a common ligand,comprising the steps of: (a) providing a plurality of samples comprisingthe protein and a first ligand under conditions wherein the first ligandand the protein form a bound complex, wherein the protein is a member ofa family of proteins that bind a common ligand; (b) assaying apopulation of candidate second ligands for the ability to transfermagnetization to the first ligand in a sample from the plurality,wherein the ability to transfer magnetization is assessed by determininga subset of magnetization signals of an isotope-edited NOESY spectrum ofthe sample; (c) identifying, from the population of candidate secondligands, a second ligand that transfers magnetization to the firstligand, thereby determining that the two ligands are proximal to eachother in a ternary bound complex with the protein; (d) observingcompetitive binding between one of the two ligands and the commonligand, thereby determining that the competitive binding ligand binds tothe common ligand binding site of the protein; and (e) obtaining apopulation of candidate binding compounds comprising the competitivebinding ligand, or a fragment thereof, linked to one of a plurality ofhomologs of the other ligand, whereby the population of candidatebinding compounds contains binding compounds that bind to members of theprotein family.

A library of second ligands can be obtained as set forth above. In thecase of an enzyme target, the library can be designed based on thestructure of a natural specificity ligand since a substrate that isacted upon by a cofactor is proximal to a common ligand, as set forthabove. A population of second ligands can also be designed to includemembers having structural features that are commonly found in aparticular class of ligands including, for example, a MOTIF library asdescribed in Example II. A population of second ligands to be used in amethod of the invention can be synthesized using combinatorial methodssimilar to those set forth above.

Thus, ligands that bind proximal to each other in a complex with amacromolecule can be identified by screening a library of candidatefirst ligands and a library of candidate second ligands.

An advantage of designing a bi-ligand binding compound based onscreening candidate ligand libraries is that the number of bi-ligandcompounds that need to be synthesized and tested compared with a classicstructure activity relationship (SAR) approach is reduced. For example,two libraries of 1000 ligands can be rapidly screened to identify asmall number of ligands that bind a macromolecule. Pairs of identifiedligands can then be combinatorially assayed for the ability tosimultaneously bind the macromolecule. If two ligands are found tosimultaneously bind the macromolecule, proximal atoms on each ligand canbe determined to guide chemistry to link the two ligands, with a smallnumber of different linkers, for example, 5. Thus, only 5 compounds needto be synthesized. In contrast, a more traditional SAR approach would beto synthesize all possible pairs, resulting in a library of about1000×1000×5=5 million compounds.

Two ligands that bind simultaneously to a macromolecule and in closeproximity to each other can be identified in a method of the inventionby detecting magnetization transfer between the two ligands when boundin a ternary complex with the macromolecule. For example, asdemonstrated in Example I, interactions between proximal ligands can beidentified based on NOE crosspeaks observed in a 2D (¹H, ¹H) NOESYspectrum. In the case of a (¹H, ¹H) NOESY spectrum obtained for ligandsin a ternary complex with a macromolecule, observation of cross peaksoccurring at the chemical shift positions of the atoms from separateligands indicate that the atoms are proximal. Such inter-ligand NOEpeaks can be resolved from intra-ligand NOE signals by adjusting themixing time in the NOESY pulse sequence. Because the strength of an NOEinteraction between two protons is dependent on 1/r⁶, where r is thedistance between the two protons, and because most inter-ligand proximalprotons will be further apart than intra-ligand proximal protons, themixing time can be increased to allow selective detection ofinter-ligand NOE peaks compared to most intra-ligand and intra-proteinNOE peaks. As another example, as demonstrated in Example VII,interactions between proximal ligands can be identified based on NOEcrosspeaks observed in a ¹³C-edited 2D NOESY spectrum. In the case of a¹³C-edited NOESY spectrum obtained for ligands in a ternary complex witha macromolecule, observations of cross peaks occurring at the chemicalshift positions of the NMR-visible atoms from separate ligands indicatethat the atoms are proximal. Alternatively, where the resonancefrequency of the protons within one ligand are sufficiently removed fromother signals of interest selective excitation, gradient sculptedexcitation with shaped pulsescan be used to excite only this resonanceof interest (see, for example, FIG. 17).

Typically magnetization transfer between proximal ligands is observed ina sample having a molar excess of ligands compared to the protein towhich they bind. Because the lifetime of an alteration to a nucleus dueto a magnetization transfer, such as an NOE interaction, is usuallylonger than the residence time for ligands in a complex with a protein,the number of ligand nuclei for which magnetization transfer is observedexceeds the number of protein molecules in the sample. Thus, the proteinacts to turn over altered ligands to amplify the observed signal in acondition where the ligand is in excess. The amplified signals can bereadily distinguished from signals arising from the protein.

In cases where ligand is not in excess over the protein, a signal thatarises from a ligand atom can be identified using known methods forassigning resonances. Such signals can be differentiated from signalsarising from other atoms in a sample using isotope enrichment. Forexample, where protons of a ligand are to be observed the protein towhich the ligand binds can be labeled with deuterium (²H) to removesignals arising from the protein. A ligand enriched with an NMRdetectable isotope at an observed atom position can be used to enhancedetection of a signal arising from the ligand atom. In addition, asignal can be selectively detected when an isotope filter or relaxationfilter is used such as any of those described in Cavanaugh et al.,Protein NMR Spectroscopy: Principles and Practice, ch. 7, AcademicPress, San Diego Calif. (1996). Examples of isotope-filtered or editedNMR methods include isolate-filtered experiments that detect 1H signalsattached to ¹²C/¹⁴N nuclei and remove ¹³C/¹⁵N-attached 1H signals, andisotope-edited (isotope-separated) experiments that detect 1H signalsattached to ¹³C/¹⁵N nuclei and remove ¹²C/¹⁴/N-attached 1H signals forselective observation of interactions between ¹³C/¹⁵N isotope-labeledand unlabeled molecules. Commonly used isotope edited or filteredmethods include 2D X-edited TOCSY, or the analogs 3D TOCSY-HMQC and 2DTOCSY-HSQC; 2D X-edited NOESY, or the analogs 3D-NOESY-HMQC and 3DNOESY-HSQC, and 3D and 4D HMQC-NOESY-HMQC. Such methods can employ avariety of well-known filters to allow the study of intramolecule NOESbetween a ligand and a protein. Exemplary filters include¹³C(ω2)-filtered/¹²C(ω1)-selected experiments and¹³C(ω1)-filtered/¹²C(ω2)-selected experiments.

The 1D(ω1)-X edited (filtered or selected) NOESY experiment produces a1D NOESY spectrum in which 1H directed attached to the X nucleic areefficiently filtered or selected before evolution during the variable t1period. 2D(ω1) filtered NOESY can be performed using presaturation,using decoupling, or using both presaturation and decoupling. Similarapproaches can be used, for example, by employing ¹³C/¹⁵N half filtersand time-shared ¹³C-filters.

To assist either rapid manual or automated analysis of spectra theω1-¹³C-filtered NOESY was recorded in the same measurement time as a 1Dexperiment (again the two IL-NOE cross-peaks are marked with the twoarrows). This simplified spectrum facilities rapid analysis bycomparison (over-lay) with 1D reference spectra of the two compounds inisolation. Upon identification of an IL-NOE, a 2D NOESY experiment canbe recorded with the same sample to characterize in detail the bindingmode of the second ligand.

A protein or'ligand-probe, such as an antenna moiety of a ligand probe,can be isotopically labeled with ²H atoms to simplify spectra byreplacing NMR-visible ¹H atoms. For example, ²H atoms can beincorporated at both exchangeable and non-exchangeable positions in amacromolecule by growing an organism expressing the macromolecule in thepresence of D₂O (²H₂O); and ²H atoms can be incorporated at bothexchangeable and non-exchangeable positions in a ligand or ligand probeby chemical synthesis methods such as those described in Example V. Aligand-probe can contain ²H atoms at positions only in an antennamoiety, at positions in the ligand, or both. ²H atoms can beincorporated or maintained at exchangeable positions, such as at amidesor hydroxyls of a protein, by carrying out steps in the isolation of themacromolecule in deuterated solvent. For protein labeling, acetate orglucose can be provided as the sole carbon source in the presence of D₂Oif complete deuteration on carbon is desired. If pyruvate is used as thesole carbon source, there will be protons only on the methyl groups ofAla, Val, Leu and Ile (Kay, Biochem. Cell Biol. 75:1-15 (1997). Forligand labeling, a variety of methods can be used, including thosedescribed in Examples VII, which show deuteration of specific positionsof small molecules.

When NOE methods are used to identify proximal ligands, the measurementscan be performed at low temperature to increase NOE build-up rates andtherefore enable the observation of inter-ligand NOES at shorter mixingtime. As temperature decreases, mixing time can be decreased resultingin (¹H, ¹H) NOESY spectra with increased sensitivity, thereby allowingobservation of peaks that are not visible or that are difficult todistinguish at higher temperatures. Furthermore, measurement ofinter-ligand NOES at shorter mixing times and lower temperature alsodecreases spin-diffusion and protein mediated magnetization transferwhich often have deleterious effects on the intensity of NOE signals. Ingeneral, higher sensitivity NOE measurements can be obtained attemperatures below 10° C. Thus, a method of the invention can includedetecting magnetization transfer at temperatures below 8° C., below 5°C. or below 2° C., so long as the sample is in a liquid state.

Further, two ligands that bind simultaneously to a macromolecule and inclose proximity to each other can be identified in a method of theinvention by detecting magnetization transfer between the two ligandswhen bound simultaneously with the macromolecule. For example,interactions between proximal ligands can be identified based onmeasuring cross-saturation between the ligands when bound in a complexwith the macromolecule. A saturation transfer difference (STD) methodcan be applied in which selective excitation of a particular resonanceof one ligand is followed by polarization transfer such thatmagnetization is effectively transferred, in a selective manner to aproximal ligand when bound in a complex. Under these conditions theresonances of non-bound molecules have an opposite sign and tend to beweaker than the resonances for bound ligands. Accordingly, STD betweenligands can be used to screen individual compounds or mixtures ofpotential ligand to identify those that bind to a target macromolecule.The cross saturation achieved via inter-ligand magnetization transfercan be achieved with natural abundance of NMR-visible isotopes. However,it can be advantageous to use deuterium labeled protein to effectivelyremove the effects of the proton mediated magnetization transfer fromthe protein. The STD method is particularly useful for use in ascreening format because the NMR signals that are used for identifyingproximal ligands can be collected on a relatively short time framecompared to other methods of determining ligand binding. In addition toproviding a functional identification that the ligand binds to theprotein, the STD method when used to identify proximal ligands providesstructural information regarding the relative location of ligands whenbound to the protein.

Because the proximity of ligands is determined based on detection ofinteractions between ligands and does not require detection ofinteractions with the macromolecule to which they are bound,isotopically labeled macromolecules are not necessary. Thus, amacromolecule used in a method of the invention can contain a naturalabundance of NMR-visible isotopes for the atoms it contains. Examples ofNMR-visible isotopes are ¹H which is present in a natural abundance of99.98%, ¹³C which is present in a natural abundance of 1.11% and ¹⁵Nwhich is present in a natural abundance of 0.37%. A macromolecule cancontain at most about 1% of the non-NMR-visible hydrogen isotope ²H, atmost 1.5% of ¹³C or at most about 0.5% of ¹⁵N.

Although labeled macromolecules are not required, a labeledmacromolecule can be used in a method of the invention. For example,once proximal ligands are identified, the orientation of one or moreligands can be confirmed or further investigated by identifying NMRinteractions with a labeled macromolecule. In applications wherelabeling of a macromolecule is desired in order to further investigatethe orientation of one or more ligand when bound to the macromolecule orto investigate structural properties of the macromolecule binding site,strategies and methods known in the art for introducing one or moreisotopic label can be used (see, for example, Laroche, et al.,Biotechnology 12:1119-1124 (1994); LeMaster Methods Enzymol. 177:23-43(1989); Muchmore et al., Methods Enzymol. 177:44-73 (1989); Reilly andFairbrother, J. Biomolecular NMR 4:459-462 (1994); Ventors et al., J.Biomol. NMR 5:339-344 (1995); and Yamazaki et al., J. Am. Chem. Soc.116:11655-11666 (1994)).

A method of the invention is well suited for use with largemacromolecules because proximal ligands in a complex with amacromolecule can be identified absent knowledge of the structure of themacromolecule or assignment of resonances for atoms of themacromolecule. In particular, large macromolecules having a monomericmolecular weight greater than 20 kDa, which often are not completely NMRassigned, or for which complete structure models are not available, canbe characterized with respect to pairs of ligands that bind thereto.Because observation of magnetization transfer between ligands can beenhanced when the ligands experience low rotational mobility,macromolecules having monomeric molecular weights greater than 25 kDa,30 kDA, 40 kDa, 50 kDa, 75 kDa, 100 kDa or 150 kDa can be used.Furthermore, a method of the invention can be used to identify proximalligands for other macromolecules with low rotational mobility such asmembrane bound proteins or multimeric proteins having at least 2, atleast 3, or at least 4 monomers, wherein the monomers can have monomericmolecular weights in the range described above.

Because structural analysis of the macromolecule itself is not requiredto identify or characterize proximal ligands in a method of theinvention, a macromolecule can be used for which resonance assignmentshave not been made for a majority of the atoms in the macromolecule.Thus, a method of the invention can use a macromolecule for which lessthan 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the atoms havebeen assigned a resonance.

Proximal ligands are identified in the methods under conditions where amajority of the macromolecule is bound in a complex with two ligands. Acondition in which a majority of the macromolecule is bound in a complexwith two ligands can be achieved when the macromolecule is present atrelatively low concentrations and excess ligand is present. Thus,although a method of the invention can be performed with millimolarconcentrations of a macromolecule, as is often required for structuredetermination by NMR, lower concentrations such as concentrations below200 micromolar can be used. The use of low concentrations of amacromolecule is advantageous when the target macromolecule is availablein limited supplies or where screening procedures require a large numberof samples containing the macromolecule. In such cases, concentrationsof the macromolecule below 100 micromolar, 50 micromolar, 40 micromolar,25 micromolar, or 10 micromolar can be used.

Ligands can be added to a macromolecule-containing sample in molarexcess such that a majority of the macromolecule in the sample will bebound in a complex with the ligands. The extent of the molar excess canbe determined using known methods for determining percent occupancybased on equilibrium binding equations, a known or predicted affinityconstant of a ligand for a macromolecule and the concentration of themacromolecule in a sample (see, for example, Segel, supra).Alternatively, excess ligands can be added and the amount sufficient toresult in a majority of the macromolecule being bound to the ligands,can be determined empirically, for example, by titration.

Proximal ligands are identified in the methods under conditions wherethe ligands bound in a complex with the macromolecule are inert tocatalysis by the macromolecule. In cases where the macromolecule is acatalyst, a ligand mimic can be chosen that does not undergo catalysisor that undergoes catalysis at a rate that is slow compared to thetimeframe in which ligand interactions are measured. In cases where areactive ligand is used with an enzyme, conversion of the ligand to aproduct can be prevented by altering conditions such that catalyticactivity of the enzyme is inhibited. For example, anaerobic conditionscan be employed to inhibit reactions requiring oxygen, pH can beadjusted to inhibit reactions requiring a particular protonation stateof a catalytic residue, or a noncompetitive inhibitor can be added.

Once a pair of proximal protons from separate ligands is identified fora particular ternary complex, the distance between the ligands can beestimated. In particular, an atom of a first ligand that is proximal toan atom of a second ligand in a ternary complex can be identified. Forexample, the distance between the ligands can be estimated based on thedistance separating the proximal protons as determined by measurementsof NOE build-up rates using methods described in Cavanaugh et al., supra(1996).

The distance determined to separate the proximal protons can then beused in combination with the average bond lengths separating otherligand atoms from the protons to estimate inter-ligand atomic distancein the ternary complex. For example, the distance between the atoms fromeach ligand that are directly bonded to the proximal protons can beestimated from the sum of the NOE measured distance and the theoreticallengths of both atom-proton bonds. Similarly, by summing the bondlengths separating other atoms from the proximal protons and consideringbond angles, the distance separating these other atoms can be estimated.Even when distances are not measured, two ligands can be identified asproximal based on observation of magnetic interactions, when spindiffusion is absent or otherwise accounted for.

Spin diffusion can be eliminated using QUIET NOESY (QuenchingUndesirable Indirect External Trouble in NOESY, Neuhaus et al. “TheNuclear Overhauser Effect in Structural and Conformational Analysis”,Wiley-VCH, New York, 2000) or NOE build-up curves. QUIET NOESYmeasurements can be performed to avoid artificial NOE cross-peaksarising from spin diffusion. These measurements differ from aconventional NOESY measurements by the presence in the middle of themixing time of a selective (or a combination of selective) 180 degreepulse(s) to invert only the signals of the two protons for which thelength of separation is to be determined. NOE build-up curves can beused to plot NOE vs. mixing time such that signals due to direct NOEtransfer can be differentiated from those that are indirect or due tospin diffusion based on the shapes of the curves as described, forexample, in Cavanaugh et al., supra (1996).

Those skilled in the art will understand that depending upon the degreeof conformational freedom for the ligands in an observed ternary complexand the number of observed inter-ligand interactions, the estimation ofdistances between atoms that are increasingly removed from the proximalligands can have different levels of precision. For example, in the caseof two proximal aromatic ring structures for which two pairs ofinteractions are observed, the relative orientations of the rings can beestimated with relatively high precision and the distance separating anyof the atoms in the two ring system can be determined with a relativelyhigh level of confidence due to two-point anchoring between the planarrings. As set forth below, the estimated distance separating two ligandscan be used to guide the selection of a linker to attach the proximalligands in designing a bi-ligand binding compound. Depending upon thelevel of confidence with which the distance is determined, the varietyof linker types and ligand attachment points represented in a library ofpotential binding compounds can be adjusted.

A ligand-probe having an attached antenna moiety can be used in a methodof the invention. An antenna moiety provides an NMR-detectable nucleusthat can occupy a position away from the ligand moiety of theligand-probe such that a magnetic interaction between the nucleus and aproximal ligand can be used to identify the relative location of theproximal ligand even if it is too distant to magnetically interact withthe ligand moiety of the ligand-probe. Thus, an antenna moiety canextend the range within which proximal ligands can be identified.

An antenna moiety used in a method of the invention can contain one ormore NMR-visible nuclei, such as one or more ¹³C, ¹⁵N, ¹⁹F, ³¹P, or¹¹³Cd. molecules. An antenna moiety also can contain one or moreNMR-invisible nuclei, such as ²H; and can contain NMR both visible andinvisible nuclei. The invention provides a compound that contains anantenna moiety useful in a method of the invention. The compound has theformula:

-   -   wherein R is any chemical moiety which binds to the        macromolecular target and where the point of attachment does not        influence binding of the ligand by methods which will be        familiar to a person skilled in the art (as judged either by NMR        direct binding, activity assay or biophysical, spectroscopic        method or computational ligand docking into a structural model).

An exemplary R group is:

A compound of the invention can be synthesized using a variety ofmethods well known to those skilled in the art. As described in ExampleVI, introduction of ¹³C carbon into the terminal methyl position byreaction of an —OH precursor ligand compound with ¹³C-methyl iodideyields the ¹³C-enriched molecule in a one-step reaction. This chemistrycan be applied to any position into which an alcohol group can beintroduced. Additionally many other high-yield routes with similarsimple one-step chemical synthesis known to those skilled in the art canbe employed, such as reaction of ¹³C-precursors with —COOH, NH2 etc.chemical groups in ligands chosen for study.

Accordingly, the invention provides a method for obtaining a focusedlibrary of candidate binding compounds for a protein family, wherein themembers of the protein family bind a common ligand. The method includesthe steps of: (a) providing a ligand-probe having an antenna moiety,wherein the ligand-probe binds to the common ligand binding site of aprotein, wherein the protein is a member of the protein family; (b)providing a sample comprising the protein, the ligand-probe and a secondligand under conditions wherein the ligand-probe, the second ligand andthe protein form a bound complex; (c) detecting a subset ofmagnetization transfer signals between antenna moiety of theligand-probe and the second ligand in the bound, wherein the signals areobtained from an isotope-edited NOESY spectrum of the sample, therebydetermining that the antenna moiety and second ligand are proximal inthe bound complex; and (d) obtaining a population of candidate bindingcompounds comprising the ligand-probe, or a fragment thereof, linked toone of a plurality of second ligand homologs, whereby the populationcontains binding compounds that bind to members of the protein family.

Also provided is a method for obtaining a focused library of candidatebinding compounds, wherein the members of the protein family bind acommon ligand. The method includes the steps of: (a) providing aligand-probe having an antenna moiety, wherein the ligand-probe binds tothe common ligand binding site of a protein, wherein the protein is amember of the protein family; (b) providing a plurality of samplescomprising the protein and the ligand-probe under conditions wherein theligand-probe and the protein form a bound complex, wherein the proteinis a member of a family of proteins that bind a common ligand; (c)assaying a population of candidate second ligands for the ability totransfer magnetization to the antenna moiety of the ligand-probe in asample from the plurality, wherein the ability to transfer magnetizationis assessed by determining a subset of magnetization signals of a anisotope-edited NOESY spectrum of the sample (d) identifying, from thepopulation of candidate second ligands, a second ligand that transfersmagnetization to the antenna moiety of the ligand-probe, therebydetermining that the two ligands are proximal to each other in a ternarybound complex with the protein; and (e) obtaining a population ofcandidate binding compounds comprising the ligand-probe, or a fragmentthereof, linked to one of a plurality of homologs of the other ligand,whereby the population of candidate binding compounds contains bindingcompounds that bind to members of the protein family.

Based on the length of an antenna moiety and its point of attachment toa ligand moiety, the relative location of a proximal ligand can bedetermined. Depending on where the antenna moiety is attached in theligand-probe, the direction and approximate location of the otherproximal ligand relative to the ligand moiety can be determined. Becausethe ligands are bound at a particular orientation in their respectivebinding sites, an antenna moiety can provide information regarding therelative structural relationships of proximal binding sites.

An antenna moiety can have any of a variety of structures that extendfrom a ligand moiety including, for example, those described below withrespect to linkers, so long as an NMR-visible nucleus is included. Anantenna moiety can have a structure that is selected based on aparticular distance desired for separating an NMR-visible nucleus andthe ligand moiety to which it is attached or based on a particularorientation for the NMR-visible nucleus relative to the ligand moiety.The relative distance and orientation can be determined based on visualinspection of a structure model for the protein to be tested or of ahomolog of the protein. The distance and orientation can also beempirically determined, for example, by iteration of a method of theinvention where the composition of the antenna moiety is altered until adesired or diagnostic interaction is observed.

An antenna moiety can have a structure that is selected based onmagnetic properties to be observed. For example, an antenna moiety cancontain an NMR-visible nucleus that is magnetically isolated from otheratoms in the ligand probe to facilitate or improve a particular NMRmeasurement. As demonstrated in Example IV use of an ether linkagefavored observation of direct NOE interactions between a terminal methyland a proximal ligand at short mixing times compared to indirect NOEinteractions due to magnetization transfer in the ligand probe. Theether linkage further provided an environment for the terminal methylprotons where relaxation effects due to vicinal proton coupling did notoccur, thereby providing a stronger signal for the methyl protons.Isolation of an NMR-visible nucleus can also be achieved by providing anadjacent ether oxygen; carbonyl carbon; thioether, sulfone or sulfoxidesulfur; deuterated carbon; selenium; or nitrogen. An NMR-visible nucleusused in an antenna moiety can be at an internal position or at aterminal position. An example of an internal position that is useful isa proton in a phenyl or other aromatic ring structure that is deuteratedat the other positions. The use of an exemplary antenna moiety having anNMR-visible ¹³C nucleus is described in Example VII.

An antenna moiety can also have an NMR-visible nucleus that is in anenvironment that differs from that of other nuclei in the ligand-probesuch that the nucleus for which observation is desired can beselectively excited. As demonstrated in Example V, methyl protons at aposition terminal to an aliphatic, ether-containing antenna moiety wereselectively saturated compared to the aromatic protons of the attachedligand moiety. Those skilled in the art will understand that antennamoieties of different lengths, composition or point of attachment can beroutinely tested using a binding assay with the target macromolecule.

A ligand-probe can contain a plurality of antenna moieties, such as 2,3, 4, 5, or more antenna moieties attached to a ligand moiety, therebyforming a ligand multi-probe. The composition, length and point ofattachment for each antenna moiety of a ligand multi-probe can bedetermined as described above. The antenna moieties included in a ligandmulti-probe can be selected such that the nuclei of each antenna that isto be observed will resonate at a frequency that is readilydistinguished from the other antenna nuclei that are to be observed.Thus, the nuclei of the antenna moieties can be separated in a singlespectrum to facilitate identification of a proximal ligand anddetermination of its orientation relative to the ligand multi-probe.

Comparison of signals arising from antenna moieties attached atdifferent points in a ligand multi-probe can provide informationregarding the direction and distance that separates it from one or moreproximal ligands. Thus, the ligand multi-probe can be used to determinethe direction and approximate location of proximal ligands bound todifferent sites on the protein. A ligand multi-probe can also be used todetermine the orientation of the ligand moiety of the ligand multi-proberelative to the proximal ligand based on the points on the ligand moietyat which each antenna moiety is attached and the atoms of the proximalligand that the antenna moiety interacts with. Thus, using the methodsdescribed below, a linker can be designed to connect the two ligands, orfragments thereof or homologs thereof, such that their relativepositions are in accordance with the observed orientations. It isunderstood that an antenna moiety can be attached to any of a variety ofligands including, for example, a common ligand or specificity ligand.

Once proximal ligands of a macromolecule are identified they can be usedto design a binding compound or a library of candidate binding compoundsfor the macromolecule. A binding compound can contain a moiety formed bya first ligand, fragment of the first ligand or homolog of the firstligand, attached by a linker to any of a second ligand, fragment of thesecond ligand or homolog of the second ligand. A fragment of a ligandincluded in a linked binding compound can be any portion of the ligandthat interacts with the target protein in such a way as to participatein specific binding. For example, a fragment of a ligand-probe that canbe linked in a binding compound can be a ligand moiety, or fragmentthereof.

A portion of a ligand that interacts with a protein can be identifiedaccording to magnetic interactions of atoms of the particular portion ofthe ligand with atoms of the protein. Such interactions can be observedusing methods known in the art such as those described in WO 99/60404and U.S. Pat. No. 5,698,401. A portion of a ligand that interacts with aprotein can also be identified by visual inspection of a structure modelof a complex of the ligand and protein, such as an X-raycrystallographic or NMR structure; docking of a structure model of theligand to a structure model of the protein; or comparison to otherligands that bind to the protein.

A library of candidate binding compounds can be obtained in which amoiety formed by a first ligand, fragment of the first ligand or homologof the first ligand is linked to a variety of homologs of the otherligand. Where the library is directed to one or more protein in a commonligand binding family, diversity of the library occurs at the portion ofthe binding compound that will interact with the specificity ligandbinding site (specificity portion), thereby providing specificity forparticular members of the family. The common ligand portion of thebi-ligand provides favorable interactions, thereby improving affinity ofthe binding compound for its target compared to the affinity that wouldbe provided by the specificity portion alone.

Diversity of the library can be further increased by using a variety oflinkers or diverse combinations of homologs of both ligands. A homologor population of homologs can be selected based on structural similarityto a particular ligand. A population of homologs can also be produced bya combinatorial approach in which a core structure of a ligand ismodified or in which moieties found in a particular ligand are combined.

A linker is selected based on the ability to provide sufficient lengthand conformational freedom for the ligands, or homologs thereof, toassociate with their respective sites on the macromolecule. A linker caninclude any number of atoms that can attain a conformation resulting inthe desired length between linked moieties including, for example, 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more atoms that are linearly connected.Linear connection is used to describe the positions of the atomsrelative to each other in a linker and is not intended to limit thelinker to a linear structure. Accordingly, a linker can have atoms thatform branch structures off of linearly connected atoms or a linker canbe formed by one or more cyclic structure.

A linker can be directly attached to a ligand at one of the atoms in theproximal pair. The linker can also be attached to a ligand, or homologsthereof, at the position of one of the atoms in the proximal pairwhether or not the same atom occupies the position in the originalligand and in the linked compound. The linker is designed to have atleast two positions for attaching at least two ligands, or homologsthereof.

The point of attachment for a linker on each ligand, or homologsthereof, can be chosen to result in a binding compound having theligands, or homologs thereof, separated by a distance similar to thatobserved for the ligands in the macromolecule complex. The distancebetween the two linked portions of a binding compound can be determinedbased on the positions of the proximal atoms in each portion. Theposition of one or both atoms can be occupied by another atom that is,for example, present due to the chemistry selected for attachment. Thedistance between the portions can also be determined based on thepositions of other atoms where the relative positions in the boundcomplex are known.

Those skilled in the art will understand that linkers of differentlengths, composition or points of attachment can be routinely testedusing a binding assay with the target macromolecule. The number ofvariations to be tested can be determined, for example, based on thedegree of confidence in the distance estimate for the two ligands to bejoined. Variations can be individually tested in a binding assay withthe target macromolecule or a library of variants can be screened forthe ability to bind to the target macromolecule. Thus, a method of theinvention can be carried out in an iterative fashion wherein the stepsof the method are repeated with linkers of different lengths orcompositions until a binding compound having a desired linkage isobtained. Similar iterations can be performed with different linkedmoieties until a binding compound having a desired affinity orspecificity or both is identified.

In another embodiment a common ligand is linked to each of a pluralityof homologs or a proximal ligand to create a focused library ofcandidate binding compounds. The use of a natural common ligand, ormimic thereof, as a partner in a linked bi-ligand can be advantageousbecause natural common ligands can be more effective in crossingbiological membranes such as bacterial or eukaryotic cell membranes. Forexample, a transport system actively transports the nicotinamidemononucleotide half of the NAD molecule (Zhu et al., J. Bacteriol.173:1311-1320 (1991)). Therefore, it is possible that a bi-ligandcomprising a common ligand, or derivative thereof, that is activelytransported into a cell will facilitate the transport of the bi-ligandacross the membrane.

Linkers that are useful for generating a binding compound include, forexample, substituted phosgene, urea, furane and salicylic acid. However,any chemical group with two reactive sites that can be used to positiona first ligand and a second ligand in an optimized position for bindingto their respective sites can be used as a linker.

Another group of linkers includes molecules containing phosphorous.These phosphorus-containing molecules include, for example, substitutedphosphate esters, phosphonates, phosphoramidates and phosphorothioates.The chemistry of substitution of phosphates is well known to thoseskilled in the art (Emsley and Hall, The Chemistry of Phosphorous:Environmental, Organic, Inorganic and Spectroscopic Aspects, Harper &Row, New York (1976); Buchwald et al., Methods Enzymol. 87:279-301(1982); Frey et al., Methods Enzymol. 87:213-235 (1982); Khan and Kirby,J. Chem. Soc. B:1172-1182 (1970)). A related category of linkersincludes phosphinic acids, phosphonamidates and phosphonates, which canfunction as transition state analogs for cleavage of peptide bonds andesters as described previously (Alexander et al., J. Am. Chem. Soc.112:933-937 (1990)). The phosphorous-containing molecules useful aslinkers can have various oxidation states, both higher and lower, whichhave been well characterized by NMR spectroscopy (Mark et al., Progressin NMR Spectroscopy 16:227-489 (1983)).

The reactive groups on a linker and the ligands, or homologs thereof, tobe attached should be reactive with each other to generate a covalentattachment of the ligands, or homologs thereof, to the linker at asufficient distance for binding to their respective binding sites on themacromolecule. A preferred reaction is that of a nucleophile reactingwith an electrophile. Many of the above described linkers haveelectrophilic groups available for attaching ligands. Electrophilicgroups useful for attaching ligands include electrophiles such ascarbonyls, alkenes, activated esters, acids and alkyl and aryl halides.

The linkers having electrophilic groups are preferably attached toligands, or homologs thereof, having nucleophilic groups including, forexample, alcohols, amines, or mercaptans. However, if a ligand, orhomolog thereof, is identified that does not have appropriate reactivegroups for attaching a linker, it can be modified to incorporate areactive group at or near the position of an atom that was identified asone of the proximal atoms. If the ligand, or homolog thereof, cannot bemodified to generate an appropriate reactive group in a desiredposition, an additional screen can be performed, as described above, toidentify a homolog having desired binding characteristics as well as achemical group in the proper position for attachment of a linker.

A compound that binds a protein can be obtained by screening a libraryof binding compounds for the ability to bind to a target macromoleculeand identifying a member of the library that binds to the protein. Thescreen can be performed using the methods described above fordetermining binding of a ligand to a macromolecule. The compound canhave specificity for a first protein over a second protein. For example,a compound can have specificity for a first protein that binds a commonligand compared to a second ligand that binds the same common ligand. Abinding compound obtained by a method of the invention can havespecificity for one or more protein of a common-ligand binding familycompared to a non-family protein. Such specificity can be due to morefavorable interactions of the specificity portion of a compound with thefirst protein compared to its interactions with the second protein.Specificity can be characterized as at least about 2 fold higheraffinity, at least about 3 fold higher affinity, at least about 4 foldhigher affinity, at least about 5 fold higher affinity, at least about10 fold higher affinity, at least about 25 fold higher affinity, atleast about 50 fold higher affinity, at least about 100 fold higheraffinity or at least about 1000 fold higher affinity.

A binding compound obtained by a method of the invention, by combiningmoieties from two ligands that bind proximal to each other in a complexwith a protein, will have higher affinity or specificity for the proteinthan the affinity or specificity of either ligand alone. The affinity ofa compound, obtained by a methods of the invention, for a protein canhave an equilibrium dissociation constant of at most about 10⁻⁶ M, 10⁻⁸M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M or 10⁻¹² M.

Although the methods of the invention have been described above withrespect to a complex in which two ligands bind a macromolecule and inwhich a bivalent binding compound is designed, a method of the inventioncan also be used to identify 3 or more ligands that are proximal whenbound to a macromolecule and to link the ligands using 2 or more linkersin order to form a multi-ligand binding compound. A method of theinvention can also be used to design a multi-ligand binding compound bysequentially adding ligands to a binding compound. Thus, a method of theinvention can include the steps of (a) obtaining a sample containing amacromolecule, a first ligand and a second ligand under conditionswherein a bound complex is formed containing the first ligand, thesecond ligand and the macromolecule; (b) detecting a subset ofmagnetization transfer signals between the first ligand and the secondligand in the bound complex, wherein the signals are obtained from anisotope edited NOESY spectrum of the sample; (c) determining from themagnetization transfer the distance between an atom of the first ligandand an atom of the second ligand in the bound complex; (d) obtaining abinding compound including the first ligand, or a fragment thereof, thesecond ligand, or a fragment thereof, and a linker, whereby the bindingcompound is capable of binding the macromolecule; and (e) repeatingsteps (b) through (d), wherein the first ligand is replaced by thebinding compound obtained in step (d) and the second ligand is replacedby another ligand.

Once a binding compound has been obtained its specificity for aparticular member of a macromolecule family can be determined bydetermining the affinity of the compound for the macromolecule comparedto other macromolecules in the family. If the compound binds to a firstmacromolecule with higher affinity or at a faster rate than a secondmacromolecule it will be identified as being specific for the firstmacromolecule. Although absence of binding to the second molecule insuch an assay is preferable in many situations, any increase inspecificity for the first macromolecule over the second can be exploitedin applications were specificity is desired. Furthermore, determinationthat a compound has specificity for one macromolecule over another, evenif moderate, can identify the compound as a candidate for iterativeimprovement in a method of the invention. In particular, the compound soidentified can be bound in a complex with the target macromolecule andused to identify a ligand that binds in a proximal location. Covalentlinkage of the compound and proximal ligand can yield a subsequentbinding compound with higher affinity and improved specificity for themacromolecule compared to other macromolecules in the same family.

The invention further provides a method for identifying a compoundhaving specificity for a particular member of a protein family, comparedto other members of the protein family, prior to synthesizing thecompound. The method includes the steps of (a) observing competitivebinding of the common ligand and a first ligand to a first protein, (b)observing competitive binding of the common ligand and a first ligand toa second protein, wherein the first and second proteins are members ofthe protein family, thereby determining that the first ligand binds tothe common ligand binding site of the first and second proteins; (c)providing a sample comprising the first protein, the first ligand and asecond ligand; (d) providing a sample comprising the second protein, thefirst ligand and the second ligand; (e) comparing the degree ofmagnetization transfer between the first ligand and the second ligandfor the samples of parts (b) and (c), wherein magnetization transfer isdetermined by detecting a subset of magnetization transfer signals froman isotope-edited NOESY spectrum of the sample; and (f) obtaining abinding compound comprising the first ligand, or a fragment thereof,linked to the second ligand, or a fragment thereof, whereby the bindingcompound selectively binds the first protein compared to the secondprotein.

Two or more ligands to be linked, or for which homologs can beidentified, in order to produce a binding compound with specificity fora first macromolecule over a second macromolecule can be identified bycomparison of magnetization transfer between the ligands when bound tothe different macromolecules. Absence of magnetization transfer betweenligands in the presence of the second macromolecule will indicate thatat least one of the ligands does not bind the second macromolecule orthat, if the ligands both bind they are relatively distal from eachother. Thus, a binding compound in which the ligands, or homologsthereof, are linked according to distances observed in the firstmacromolecule will have reduced affinity for the second macromoleculeeither because one of the ligands, or homologs thereof, does notcontribute to a favorable binding interaction or because the ligands, orhomologs thereof, are sterically constrained from binding to both siteson the second macromolecule. Similarly, a library of candidate bindingcompounds will have a higher probability of containing a compound thatis specific for the first macromolecule.

A greater degree of magnetization transfer between two ligands whenbound to a first macromolecule compared to when bound to a secondmacromolecule can indicate a shorter distance between the ligands in thefirst macromolecule. Based on the distance measured between the ligandsin both macromolecule complexes the length of a linker can be chosen tofavor binding to the first macromolecule by being long enough to allowthe two ligands, or homologs thereof, to bind to both sites on the firstmacromolecule but too short to allow both ligands, or homologs thereof,to bind their respective sites on the second macromolecule.

The following examples are intended to illustrate but not limit thepresent invention.

Example I Design of a Potent and Specific Bi-Ligand for P38α Map Kinase

This Example demonstrates use of the methods of the invention to designa potent and selective inhibitor for activated p38α MAP kinase, startingfrom relatively weak binding fragments.

A library of 29 PBBA structure analogs was screened against unlabeledp38α MAP kinase (p38α) as follows. Samples containing 10 to 30micromolar concentration of unactivated p38α MAP kinase and 0.1 to 1.0millimolar concentration of one of the PBBA structure analogs wereobtained. The samples were screened using WaterLOGSY with saturationstimes of 2 s (frequency selective excitation via a train of 232 6 ms itpulses with a GAUSS profile at an 80 Hz RF field strength) with solventsuspension using WATERGATE at 4° C. WaterLOGSY is further described inDalvit et al. J. Biomol. NMR 21:349-59 (2001) Dalvit et al. J. Magn.Res. B112:282-288 (1996) and Dalvit et al., J. Biomol. NMR 11:437-444(1998). Among the compounds screened, p-butyl benzoic acid (PBBA) showedcross-saturation effects with p38α indicating binding to the protein.

Potential inhibitors designed to mimic the natural cofactor, asdetermined by visual inspection of commercially available compounds andassessment of the scientific literature pertaining to kinase medicinalchemistry, were screened against a complex of p38α and PBBA as follows.Samples containing 10 to 30 micromolar concentration of unactivated p38αMAP kinase and 0.1 to 1.0 millimolar concentration of PBBA and 0.1 to1.0 millimolar concentration of one of the potential inhibitors wereobtained. For each sample a 2D [¹H, ¹H] NOESY experiment was performedat 4° C. This process identified 7 molecules that bound proximal to theterminal methyl of PBBA. FIG. 2 shows the structure of PBBA, with theterminal methyl represented by an asterisk and structures of the 7molecules where arrows indicate the regions of each molecule thatcontained atoms having NOE interactions with the terminal methyl ofPBBA.

FIG. 3 shows exemplary NMR NOESY data for the ternary complex formed byp38α, PBBA and the TTM2001.082.B09 inhibitor molecule shown in the firstpanel of FIG. 2 a. As shown in FIG. 3, NOE crosspeaks were observed foratoms of the aliphatic moiety of PBBA with atoms of the fluoro-phenyl ofthe inhibitor TTM2001.082.B09. The crosspeaks indicated that: the atomsof the aliphatic moiety of PBBA, identified as atoms 1 to 4 in the righthand panel of FIG. 3, were proximal to the atoms of the fluoro-phenylmoiety of the inhibitor TTM2001.082.B09 that are labeled as atoms a andb in the right hand panel of FIG. 3.

NOE buildup experiments were performed on the sample containing p38α MAPkinase, PBBA and the inhibitor TTM2001.082.B09 and used to determine thedistance between atoms 1 to 4 of PBBA and atoms a and b of the inhibitorTTM2001.082.B09. Based on the NOE buildup experiments, the distancebetween the terminal methyl of PBBA and atom a of the inhibitorTTM2001.082.B09 was determined to be 0.5 Å.

The TTM2001.101.A09 bi-ligand compound (shown in FIG. 4) was designedbased on the NOE determined distances to contain a moiety similar to thePBBA molecule and a moiety similar to the inhibitor TTM2001.082.B09joined by a thioether (—CH₂—CH₂—S—CH₂—) linker. The TTM2001.101.A09bi-ligand compound was synthesized as follows.4-(6-(Acetylsulfanyl)hexyl) benzoic acid methyl ester (0.985 mmol) wasdeprotected, removing the thioacetate group, in a biphasic mixturecontaining potassium carbonate (4.34 mmol) in nitrogen-purged methanol(4 ml), water (2 ml) and tetrahydrofuran (2 ml) that was stirred at roomtemperature for 1.5 hrs under nitrogen. Esterified TTM2001.101.A09(1-(4-Fluoro-3-(6-((4-methoxycarbonyl-phenyl)hexylsulfanyl)methyl)phenyl)-1H-benzoimidazole-5-carboxylicacid) was synthesized by then adding1-(3-Chloromethyl-4-fluor-phenyl)-1H-benzimidazole-5-carboxylic acid(0.820 mmol) to the stirred deprotection mixture. Following work up byremoving volatile solvent in vacuo, diluting with water, acidifying with2N HCl to pH <1, diluting with brine and extracting with ethyl acetate,the esterified TTM2001.101.A09 product was purified with flashchromatography (gradient 95:5 dichloromethane/methanol to 90:10dichloromethane/methanol). The Ester was removed from the purifiedproduct by stirring at room temperature for 15 hours with lithiumhydroxide (1.25 mmol) in methanol (1 ml) and water (1 ml), followed byaddition of another 1.25 mmol of lithium hydroxide hydroxide andstirring at room temperature for another 24 hours. TTM2001.101.A09 waspurified from the mixture by acidification with 2N HCl until pH <2 andcollection of the white precipitate by filtration and washing with waterand ether.

The ability of TTM2001.101.A09 to bind to p38α was determined bycomparing the degree of line broadening in 1D ¹H NMR spectra for thecompound in the presence of p38α compared to in the absence of p38α. Asshown in FIG. 4, significant line-broadening was observed in the 1D ¹HNMR spectrum of 50 micromolar of TTM2001.101.A09 in the presence of 10mM of p38α. (FIG. 4 b) compared to the spectrum obtained for 50micromolar of TTM2001.101.A09 in the absence of p38α (FIG. 4 a),indicative of tight binding between p38α and TTM2001.101.A09.

The TTM2001.101.A09 bi-ligand compound and the fragments from which itwas constructed were tested as inhibitors of p38α enzymatic activity inan assay measuring phosphorylation of myelin basic protein (MBP) byp38α. The results of the assay are shown in FIG. 5, and indicated thatTTM2001.101.A09 bound to p38α with an IC₅₀ of 1.7 micromolar, which wasgreater than 100 fold tighter than either of the starting fragments.Correction of the IC₅₀ of TTM2001.101.A09 for high ATP concentrationindicated that the K_(d) for binding between p38α and TTM2001.101.A09was about 300 nanomolar.

Example II Combinatorial Matching of Fragments with the MOTIF Library

This example demonstrates the creation of a library of molecules, termeda MOTIF library, having sub-structural features or moieties that arecommonly found in marketed drugs or other compounds that have beenevaluated in a clinical setting. This example further describes the useof NMR ACE to screen a MOTIF library to obtain a bi-ligand thatspecifically binds a protein.

A number of PBBA related compounds were screened with WaterLOGSY toidentify those that bound to p38α. WaterLOGSY screening identified theligands shown in FIG. 6. Of the compounds shown in FIG. 6, two wereidentified by cross-saturation experiments (with 10 μM unactivated p38α,1.5 s to 3.0 saturation with a train of IBURP pulses) to interact withTTM2001.082.A10 as indicated by the arrows.

A library, referred to as the MOTIF library, containing molecules havingsub-structural features or moieties that are commonly found in marketeddrugs, as well as other compounds that have been evaluated in a clinicalsetting was constructed as follows. A database of compounds that areeither marketed as drugs or that have undergone clinical trials wascreated. The database contained over 3500 different chemical entities.The prevalence of particular fragments in the database was analyzed. Forexample, the diphenyl amine moiety was found in 96 out of 3882compounds, or 2.5% of the compounds. Moreover, this moiety was found inmultiple pharmacological classes. Thus, diphenylamine was identified asa small molecular weight molecule that can be used as part of ascreening library for NMR ACE.

Small molecules that contain the diphenyl amine moiety were included inthe MOTIF library. Other criteria for determining whether a molecule wasto be included in the MOTIF library included a maximum molecular weightof 220 Daltons and chemical inertness under the assay conditions. Basedon these criteria a diverse MOTIF library of 160 small drug-likecompound fragments was arrayed in multiwell plates for screening.

The MOTIF library members were characterized in terms of solubility and1D and 2D COSY NMR spectroscopy to obtain proton assignments. Themembers of the MOTIF library that were found to have favorablecharacteristics such as solubility and resolved assignable protons werethen screened against samples containing a complex of p38α and one ofthe 6 PBBA-related compounds shown in FIG. 6. The screen was repeatedsuch that each member of the MOTIF library, that was found to havefavorable characteristics, was screened against six different samplescontaining p38α and, respectively, each of the PBBA-related compoundsshown in FIG. 6. Samples found to contain ternary complexes were furtheranalyzed by NOE buildup experiments to determine distances between pairsof MOTIF library members and PBBA-related compounds in the ternarycomplexes. One pair of ligands that bound proximal to each other withp38α was TTM2001.082.A10 and TTE2001.084.47A.

Bi-ligand binding compounds are synthesized to have covalently attachedmoieties based on the chemical identities of and distances between thepairs of MOTIF library members and PBBA-related compounds that are foundto be proximal in the ternary complexes.

Example III Gene Family Focused Libraries with NMR ACE

This example demonstrates the use of competition experiments andstructure analysis combined with NMR ACE to design focused librariestargeted to a particular protein or family of proteins.

Using the methods described in Example I, PBBA and SB203580 were foundto bind to p38α to form a ternary complex where PBBA was proximal toSB203580. As shown in the upper panel FIG. 7, NOE interactions wereobserved between atom 4 of PBBA and atoms a and b of SB203580. Based onNOE buildup experiments the distance between atom 4 of PBBA and atom bof SB203580 was determined to be 3 0.5 Å. Thus, PBBA bound to p38α at asite that was proximal to the ATP common ligand binding site.

Specificity Pocket

The location where PBBA binds to p38α was predicted as follows. SB203580is a known inhibitor of p38α that shows competitive binding with ATP inenzymatic assays, indicating that it binds in the ATP site. Binding ofSB203580 to the ATP site of p38α has also been observed with a crystalstructure of the SB203580/p38α complex shown in the lower panel of FIG.7 (see Wang et al. Structure 6:1117-1128 (1998) and Protein Data Bankentry 1BL6.pdb). Cross-saturation competition studies indicated thatSB203580 was not able to displace PBBA. Furthermore, PBBA was not ableto displace a fluorescently tagged staurosporine in a fluorescencepolarization experiment. Staurosporine is known to bind in the ATP siteof p38α. Thus, both NMR and traditional displacement experimentsindicated that PBBA did not bind in the ATP site.

A peptide having the sequence IPTTPITTTYFFFKKK (SEQ ID NO:1) is a knownphosphorylation substrate for p38 as described, for example, in Chen etal. Biochemistry 39:2079-2087 (2000). This peptide could not displacePBBA from p38α in WaterLOGSY competition experiments, indicating thatthe peptide and PBBA occupied different binding sites on p38α. However,PBBA was shown to inhibit phosphorylation of MBP protein by p38α asdescribed in Example I and shown in FIG. 5. Since the Mbp binding siteincludes, but extends beyond the peptide binding site, these variouscompetition experiments suggest that PBBA binds in a part of the MBPsubstrate binding site that extends beyond the peptide binding site.This site is shown in the lower panel of FIG. 7, and is referred to asthe SL (specificity ligand) site, since extending a bi-ligand libraryinto this site off of an ATP mimic might provide additional specificity.

Based on the distances determined between PBBA and SB203580, thelocation where SB203580 binds to p38α, and the results of thecompetitive binding assays described above, the relative locations ofthe binding regions of p38α for ATP, MBP, the peptide of SEQ ID NO:1 andPBBA were determined. A schematic diagram showing the relative locationsof these binding regions is provided in FIG. 8. As shown in FIG. 8, theATP binding region (dark shaded region) is adjacent to the MBP bindingregion (white region) and within the MBP region is a region where PBBAbinds (indicated by brackets) as well as a region where the peptide (SEQID NO:1, lightly shaded region) binds. As shown in FIG. 8, the region ofp38α where PBBA binds is separate from the region of p38α wherephosphorylation occurs.

The locations of the binding regions of p38α for ATP, Mbp, the peptideof SEQ ID NO:1 and PBBA were further defined based on a structuralcomparison of p38α-like proteins as follows. FIG. 9A shows a portion ofthe model of the p38α structure from Wang et al. supra (1998)(ProteinData Bank entry 1BL6.pdb) which includes the regions diagramed in FIG. 8and where residues are color coded based on the degree of conservationbetween the residues of the p38α-like proteins. The degree ofconservation was determined using PrISM (Yang and Honig Proteins37:66-72 (1999)) and Psi-blast (Altschul et al., Nucleic Acids Res.25:3389-3402 (1997)). Residue conservation scores were obtained from themultiple structure and sequence alignments, which range from highlyhomologous (blue) to distantly related (red) residues. As shown in FIG.9, the region of p38α that binds PBBA is variable. Because the regionwhere PBBA binds is variable it is termed a specificity ligand site ofp38α. The ATP binding site has a conserved structure and is referred toas a common ligand site of the p38α-like proteins.

The peptide (SEQ ID NO:1) was modeled into the p38α structure based onits location in the PKC crystal structures described in Nishikawa et al.J. Biol. Chem. 272:952-960 (1997); Nair et al. J. Med. Chem.38:4276-4283 (1995); Songyang et al. Cur. Biol. 4:973-982 (1994);Songyang et al. Mol. and Cell. Biol. 16:6486-6493 (1996). Incorporatingresidue conservation scores as the starting point, computational dockingsimulations were performed with small molecules using GOLD and otherknown methods as described, for example, in Doucet and Weber,“Computer-Aided Drug Design” Academic Press (1996). The simulationsshowed that molecules, such as PBBA, docked into the specificity regionindicated by the circle in FIG. 9B. The location of the SB203580 ATPmimic is represented with a pentagon in FIG. 9 and the relationshipbetween PBBA and SB203580 is indicated with arrows. The arrows span thecone of area that can be occupied by the benzoic acid moiety of PBBA,when constrained to have the terminal methyl proximal to SB203580according to the NOE of FIG. 7. The electrostatic surface potential mapof p38α was calculated using the Grasp algorithm (the Grasp algorithm isdescribed, for example, in Nicholl et al. Proteins: Strut. Func. andGenet., 11:281-296, (1991)).

Design of a Focused Library

Based on the results described above, the region of p38α that binds PBBAis predicted to be a target for binding compounds having specificity fora particular member of this gene sub-family. The proximity of thisspecificity ligand site to the relatively conserved ATP site indicatesthat a bi-ligand library can be constructed in which a common ATP orATP-like moiety is linked to one of a variety of moieties that aresimilar to PBBA or that bind to the same site as PBBA. A moiety thatbinds to the same site as PBBA is determined by docking a model of themoiety to the PBBA binding site, by structural comparison to PBBA or byidentifying ligands that bind to p38α in an in vitro binding assay.

In order to create a focused library that is specific to p38α andrelated protein kinases, moieties can be chosen based on specificity forthe PBBA binding site of p38α compared to other p38α-like proteins.

Example IV Identifying Ligand Location with a Ligand-Probe Containing anAntenna Moiety

This example demonstrates the use of a common ligand-probe having anantenna moiety capable of detecting a proximal second ligand. Thisexample further demonstrates discrimination of the relative position andorientation of second ligands using a common ligand-probe having anantenna moiety.

Ligand-probe TTM2002.143.A27 contains an ATP mimic core moietycovalently attached via an amine linkage to a 3-oxabutyl antenna moietyas shown in FIG. 10. The TTM2002.143.A27 ligand-probe was designed basedon the binding orientation and position of the parental common ligand ina 3-dimensional structure of the p38α protein kinase. The antenna moietywas placed such that it can extend from the core structure toward aproximal binding site.

The ether linkage in the antenna moiety allows the terminal methyl groupto be relatively isolated from the other protons in the ligand probe,thereby favoring observation of direct NOE transfer from the methyl to aproximal ligand. The ether linkage allows greater differentiation ofdirect NOE interactions between the methyl and a proximal ligandcompared to indirect NOE interactions from a proximal ligand through thecore moiety of the ligand probe to the methyl. Isolation of the methylgroup due to distance from the other protons allows direct NOE transferto be selectively observed by obtaining spectra at relatively shortmixing times. Furthermore, the absence of vicinal protons minimizesrelaxation effects for the methyl protons, thereby providing a strongersignal.

From the MOTIF library, described in Example II, 25 compounds wereidentified that bound to the p38α protein kinase at sites different fromthe common ligand. These 25 compounds were screened for proximal bindingnear the core of the parental common ligand as follows. Samples wereobtained containing 100 to 1000 micromolar concentrations of theTTM2002.143.A27 ligand-probe, 10 to 50 micromolar concentrations ofactivated p38α protein kinase, and one of the 25 compounds at aconcentration of 100 to 1000 micromolar. The samples were screened forproximal ligand interactions by (¹H, ¹H) 2D NOESY acquisitions usingmixing times of 100-1200 msec at 4° C.

Among the 25 compounds screened, p-chloro-phenol (PCP, TTE0020.003.A05)exhibited NOEs to protons located within the antenna moiety of theTTM2002.143.A27 ligand-probe. As summarized in FIG. 10, for thep38α-TTM2002.143.A27-PCP ternary complex, NOEs were identified betweenthe aromatic hydrogen protons of PCP and the aliphatic protons in theantenna of TTM2002.143.A27. No NOE crosspeaks of significant intensitywere observed from PCP to aromatic protons in the core of the parentalcommon ligand in NOESY spectra. Thus, the binding site of PCP appearedto be restricted to a location on the surface of p38α that was within6.0 Å of the antenna moiety, but at a distance greater than 6.0 Å fromthe aryl rings in the core moiety of TTM2002.143.A27. Inter-liganddistance between the PCP ligand and antenna moiety are determined basedon inspection of the build-up of intensity in NOESY interactions as afunction of mixing time (τ_(m)).

Also among the 25 compounds screened, the TTE0020.003.A09 ligand wasfound to bind proximal to the TTM2002.143.A27 ligand-probe. Assummarized in FIG. 11, NOE cross peaks were observed between aliphaticprotons of the TTE0020.003.A09 second ligand and the core moiety of theligand probe. However, no significant inter-ligand NOE cross-peaks wereobserved to the antenna-probe. Thus, the binding site of TTE0020.003.A09appeared to be at a location of p38α that was within 6.0 Å of the arylrings in the core moiety of TTM2002.143.A27, but at a distance greaterthan 6.0 Å from the antenna moiety.

Comparison of the NOEs observed between the ligand-probe and PCP withthe NOEs observed between the ligand-probe and TTE0020.003.A09,indicates that addition of the antenna moiety provides informationdiscriminating between the locations of the differing binding sites forsecond ligands.

Example V Identification of Proximal Ligands by Selective CrossSaturation of an Antenna Moiety

This example demonstrates identification of proximal ligands usingselective cross saturation of protons of an antenna moiety attached to aligand probe.

Ligand-probe TTM2002.143.A27 was obtained as described in Example IV.The protons of the terminal methyl group of the antenna moiety can beselectively saturated compared to other protons of the ligand probebecause the methyl is isolated by the adjacent ether group and becausethe frequency of saturation, for the methyl protons is different fromthat of the aromatic ring protons.

The 25 compounds from the MOTIF library described in Example IV werescreened for proximal binding near the ligand-probe as follows. Sampleswere obtained containing 100 to 1000 micromolar concentrations of theTTM2002.143.A27 ligand-probe, 10 to 50 micromolar concentrations ofactivated p38α protein kinase, and one of the 25 compounds at aconcentration of 100 to 1000 micromolar. Samples were screened for thepresence of second ligands binding proximal to the common ligand-probeby (¹H) 1 dimensional saturation transfer difference experiments usingsaturations times of 2 s (frequency selective excitation via a train of232 6 ms π pulses with a GAUSS profile at an 80 Hz RF field strength)with solvent suspension using WATERGATE at 4° C.

Among the compounds screened, the aromatic protons of p-pentyl-aniline(PPA) showed a reduction in the intensity on selective saturation of themethyl protons located within the terminus of the antenna-probe (2 ssaturation at 3.0 ppm), relative to a control experiment (withoff-resonance saturation 5000 Hz up-field). As summarized in FIG. 12intensity changes were observed for two aromatic protons in a secondligand (TTE0020.003.A09) indicating that they were proximal to theantenna probe when bound to p38α.

These results demonstrate that proximally bound ligands can beidentified by observing reduced intensity of resonances for protons in asecond ligand that bind close to an antenna moiety for which the protonresonances have been selectively saturated. Such one-dimensional NMRexperiments can be performed in minutes, allowing a roughly ten-foldreduction in the screening time per compound compared to 2D NOESY basedmethods. Such a pre-selection approach can be applied prior to detailedcharacterization by 2D (¹H, ¹H) NOESY or as an alternative to 2Dapproaches, to increase screening throughput and reduce instrumentationdemands.

Example VI Use of Antenna Distance Measurements to Guide Linkage of F1and F2 Fragments

This example describes the use of antenna moiety distance measurementsfor determining the linkage of a specificity ligand, or portion thereof,also referred to as an F₁ fragment, and a common ligand, or portionthereof, also referred to as an F2 fragment, and shows that deuterationof CH2 groups in the antenna moiety advantageously allows acquisition ofaccurate distance measurements.

Distance measurements from an NMR-visible nucleus of an antenna moietyto common ligand and specificity ligand portions of a binding compoundwere used for guiding rational compound design. FIG. 18 shows arepresentation of the distances between the specificity ligand fragment,termed F1, and the common ligand fragment, termed F2.

Using 1H-1H NOES intensity measurement, spin diffusion effects can maskthe difference in distances measured between moieties of a ligand-probe.For example, the methyl-group at the terminus of the antenna moiety(distance A) and the proximal F2 fragment and the protons with the coreof the F1 fragment (distance B) of the ligand probe shown in FIG. 18 aresubject to spin diffusion effects. Specifically, spin diffusionprocesses lead to similar NOE intensities for CH₃->F1 core NOES andCH₃->F2 (4-chlorophenol) NOES. This effect is seen in the results of anexperiment shown in FIG. 19. The experiment was a 300 ms 2D NOESY using39 μM Protein activated human p38α kinase, in 50 mM Potassium Phosphatebuffer pH 7.6; 20% H2O/80% D₂O with ligand concentrations of 500 μM at277K.

To remove these spin diffusion effects, the two CH2 groups in theantenna moiety were deuterated, and NOESY of the binding compound in thepresence of p38α was performed. The experiment was a 300 ms 2D NOESYusing 39 μM Protein activated human p38α kinase, in 50 mM PotassiumPhosphate buffer pH 7.6; 20% H2O/80% D₂O with ligand concentrations of500 μM at 277K. Results from this experiment are shown in FIG. 20.

Deuteration of the two CH2 groups in the antenna moiety removes the spindiffusion pathway through the ligand, although that through the proteinremains, and the distances extracted from the NOESY spectrumconsequently reflect the true distances more faithfully. IL-NOE crosspeaks in the row are marked with arrows in FIG. 19.

Thus, distance measurements between an antenna moiety and other portionsof a binding compound can be accurately determined using an antennamoiety in which 1H atoms have been replaced by 2H atoms.

Example VII Simplification of IL-NOE Spectra Using Isotope-Editing

This example shows simplification of IL-NOE spectra usingisotope-editing methods.

2D spectra are highly complex and are difficult to interpret eithermanually or automatically (see, for example, FIG. 15, right panel). Tosimplify analysis of 2D spectra, isotope-editing methods can be used.For example, using these methods, the identification of proximal F2binders to the CH3 group in the terminus of the antenna moiety to aregion of the protein targeted for “scanning/probing” (two NOEcross-peaks indicated by arrows in the right panel) was performed usingonly one row of signals.

A ¹³C carbon was introduced into the terminal methyl position byreaction of an —OH precursor F1-compound with ¹³C-methyl iodide. Withthis ¹³C-enriched terminal methyl-group and the introduction of a¹³C-half filter at the beginning of the 2D Inter-Ligand NOESY (IL-NOESY)experiment the spectrum was simplified dramatically to a single row ofNOE cross-peaks which contain relative distance and orientationinformation describing the binding of a second fragment in relation tothe position of the terminal methyl group in the antenna moiety. FIG. 15shows ¹³C-filtered 2D NOESY, left panel, inter-ligand NOE cross withinthis single row marked with arrows.

To assist either rapid manual or automated analysis of spectra theω1-¹³C-filtered NOESY was recorded in the same measurement time as a 1Dexperiment (again the two IL-NOE cross-peaks are marked with the twoarrows). This simplified spectrum facilities rapid analysis bycomparison (over-lay) with 1D reference spectra of the two compounds inisolation. Upon identification of an IL-NOE, a 2D NOESY experiment canbe recorded with the same sample to characterize in detail the bindingmode of the second ligand.

Experiments shown in FIG. 16 are 300 ms 2D NOESYs using 39 μM Proteinactivated human p38α kinase, in 50 mM Potassium Phosphate buffer pH 7.6;20% H2O/80% D₂O with ligand concentrations of 500 μM at 277K with a 4hour acquisition time. The experiment shown in FIG. 17 is a 300 ms 1DNOESY using 12 μM Protein activated human p38α kinase, in 50 mMPotassium Phosphate buffer pH 7.6; 20% H2O/80% D₂O with ligandconcentrations of 500 μM at 277K with a 4 hour acquisition time. Theshaped pulse was a 40 ms pulse with a truncated Gaussian profile.

Example VIII Synthesis of a Ligand-Probe for Isotope-Edited NMR Assemblyof Chemical Entities

This example shows synthesis of a ligand probe useful in the methods ofthe inventions.

To a solution of 4-Picoline (9.3 g, 0.10 mol) and ethyl 4-fluorobenzoate(16.8 g, 0.10 mol.) in anhydrous THF (300 mL) was added potassiumbis(trimethylsilyl)amide (KHMDS, 20 g, 0.10 mmol) at 0° C. The reactionmixture was warmed up to room temperature and stirred for overnight atroom temperature. The reaction product of yellow precipitate wasfiltered and washed with THF. Additionally, the filtrate wasconcentrated then water was added. More yellow crude product was formedupon addition of water. The filtrate was then extracted with ethylacetate. The organic layer was dried over MgSO₄, and concentrated togive more products. The total amount of product is 15.0 g (71%). ¹H NMR(300 MHz, DMSO-d6) δ 4.49 (s, 2H, CH₂CO—), 7.29 (d, J=5.1 Hz, 2H, 2 ═CHof Py), 7.39 (dd, J₁=J₂=8.3 Hz, 2H, 2 ═CH of FPh), 8.14 (dd, J₁=8.3 Hz,J₂=5.6 Hz, 2H, 2 ═CH of FPh), 8.51 (d, J=5.1 Hz, 2H, 2 ═CH of Py) ppm

The mixture of 4-fluorobenzoyl-4′-pyridyl methane (11.84 g, 55.1 mmol)and hydroxyethyl hydrazine (90%, 4.5 mL, 59.5 mmol) in ethyl alcohol (50mL) containing acetic acid (0.5 mL) was refluxed for 2 h, then cooled toroom temperature. Dimethylforamide dimethylacetal (DMFDMA, 27 mL, 202mmol) was added and the reaction mixture was refluxed overnight. Thereaction was cooled down to room temperature and concentrated to removeethyl alcohol. Water was then added and pale yellow solid was formed.The solid was filtered and washed with 10% ethanol-water, the paleyellow solid was dried and obtained as the correct product. Thefilterate was concentrated and more products were precipitated out. Thetotal amount was 15.2 g (97%). ¹H NMR (300 MHz, DMSO-d6) δ 3.85 (m, 2H,—CH₂OH), 4.25 (t, J=5.4 Hz, 2H, —CH₂N═); 5.06 (t, J=5.4 Hz, 1H, —OH),7.24 (m, 4H, 2 ═CH of Py & 2 ═CH of FPh), 7.47 (dd, J₁=8.8 Hz, J₂=5.8Hz, 2H, 2 ═CH of FPh), 8.23 (s, 1H, 1H of pyrazole), 8.50 (d, J=4.6 Hz,2H, 2 ═CH of Py) ppm. FAB-MS (m/z): 284.27 (M+1, 100).

To a solution of3-(4-Fluorophenyl)-4-(4-pyridinyl)-1H-pyrazole-1-ethanol (0.20 g, 0.72mmol) in anhydrous THF (15 mL) was added NaH (56 mg, 2.33 mmol) followedby the addition of methyl iodide (45 μL, 0.74 mmol). The reactionmixture was stirred overnight. Water was added to quench the reactionand the reaction mixture was extracted with dichloromethane (2×30 mL).The organic layer was dried over MgSO₄, filtered and concentrated togive the crude product which was purified by silica gel chromatography(eluent: 20/1 CH₂Cl₂/MeOH) to give the product (0.12 g, 57%) as paleyellow solid. ¹H NMR (300 MHz, CDCl₃) δ 3.40 (s, 3H, —OMe), 3.82 (t,J=4.4 Hz, 2H, —CH₂N═), 4.40 (t, J=4.4 Hz, 2H, —CH₂OCH₃), 7.15 (dd,J₁=J₂=8.3 Hz, 2H, 2 ═CH of FPh, 7.42 (dd, J₁=8.3 Hz, J₂=5.3 Hz, 2H, 2═CH of FPh), 7.66 (brs, 2H, 2 ═CH of Py), 8.03 (s, 1H, 1H of pyrazole),8.63 (brs, 2H, 2 ═CH of Py) ppm. FAB-MS (m/z): 298.3 (M+1, 100).

To a solution of3-(4-Fluorophenyl)-4-(4-pyridinyl)-1H-pyrazole-1-ethanol (0.20 g, 0.72mmol) in anhydrous THF (15 mL) was added NaH (56 mg, 2.33 mmol) followedby the addition of ¹³CH₃I (45 μL, 0.74 mmol). The reaction mixture wasstirred overnight. Water was added to quench the reaction and thereaction mixture was extracted with dichloromethane (2×30 mL). Theorganic layer was dried over MgSO₄, filtered and concentrated to givethe crude product which was purified by silica gel chromatography(eluent: 20/1 CH₂Cl₂/MeOH) to give the product (0.11 g, 52%) as paleyellow solid. ¹H NMR (300 MHz, CDCl₃) δ 3.42 (d, J=141.6 Hz, 3H, —OMe),3.85 (m, 2H, —CH₂N═), 4.39 (t, J=5.0 Hz, 2H, —CH₂OCH₃), 7.08 (dd,J₁=J₂=8.6 Hz, 2H, 2 ═CH of FPh, 7.20 (d, 2H, J=4.6 Hz, 2 ═CH of Py),7.49 (dd, J₁=8.6 Hz, J₂=5.4 Hz, 2H, 2 ═CH of FPh), 7.75 (s, 1H, 1H ofpyrazole), 8.51 (brs, 2H, 2 ═CH of Py) ppm. FAB-MS (m/z): 299.3 (M+1,100).

To a solution of ethylene glycol-d₆ (1.00 g, 14.7 mmol) in 20 mL ofmethylene chloride was added tert-butyldimethylsilyl chloride (2.21 g,14.7 mmol). The solution was stirred at room temperature for 3 h andwashed with water (2×5 mL). The organic phase was dried and concentratedto dryness. Chromatography (EtOAc/hexane 1:7) gave 1.16 g (44.8% yield)of title compound. ¹H NMR (CDCl₃): δ 0.93 (s, 9H), 0.12 (s, 6H) ppm.

To a solution of compound 2 (1.600 g, 9.1 mmol), Et₃N (1.840 g, 22.8mmol) and 4-dimethylaminopyridine (20 mg) in 15 mL of methylene chloridewas added toluenesulfonyl anhydride (5.000 g, 15.2 mmol). The solutionwas stirred overnight and concentrated. Chromatography (EtOAc/hexane1:10) gave 2.75 g (91.4% yield) of tosylate compound. ¹H NMR (CDCl₃): δ7.84 (d, J=7.9 Hz, 2H), 7.37 (d, J=7.9 Hz, 1H), 2.49 (s, 3H), 0.90 (s,9H), 0.08 (s, 6H) ppm.

A solution of 3-(4-fluorophenyl)-4-(pyridin-4-yl)pyrazole (0.600 g, 2.5mmol) and NaH (0.180 g, 7.5 mmol) was heated to 70° C. for 0.5 h,followed by addition of a solution of compound 3 in 3 mL of DMF. Thesolution was kept at 70° C. for 3 h, then diluted with EtOAc (50 mL) andwashed with water. The separated organic phase was dried andconcentrated. Chromatography (EtOAc/hexane 3:1) gave 0.250 g (24.9%) oftitle compound. ¹H NMR (CDCl₃): δ 8.49 (d, J=5.2 Hz, 2H), 7.76 (s, 1H),7.41 (m, 2H), 7.25 (m, 2H), 7.07 (t, J=8.6 Hz, 2H), 0.83 (s, 9H), −0.02(s, 6H) ppm; ¹³C NMR (CDCl₃): δ 164.02, 160.74, 149.69, 148.39, 140.86,131.08, 130.02, 129.92, 129.01, 128.97, 122.19, 121.23, 117.19, 115.36,115.08, 25.54, 17.99 ppm; MS (M+1)⁺ 402.

A mixture of compound 5 (0.25 g, 0.62 mmol) and 1 M solution oftetrabutylammonium fluoride (0.7 mL) was stirred for 0.5 h.Concentration and chromatography (EtOAc/hexane 10:1) gave 0.168 g (94.5%yield) of title compound. ¹H NMR (CDCl₃): δ 8.54 (d, J=5.0 Hz, 2H), 7.80(s, 1H), 7.41 (m, 2H), 7.29 (d, J=5.0 Hz, 2H), 7.12 (t, J=8.5 Hz, 2H)ppm; ¹³C NMR (CDCl₃): δ 164.21, 160.93, 149.32, 148.53, 141.19, 130.94,130.12, 130.02, 128.73, 128.69, 122.38, 117.26, 115.58, 115.29 ppm; MS(M+1)⁺ 288.

To a solution of compound 6 (0.040 g, 0.14 mmol) and NaH (0.010 g, 0.42mmol) in 5 mL of DMF was added a solution of iodomethane (0.029 g, 0.21mmol) in 0.5 mL of DMF. The solution was stirred for 2 h andconcentrated. The resultant residue was dissolved in 30 mL of EtOAc andwashed with water (5 mL). The organic phase was dried and concentrated.Chromatography (EtOAc/hexane 10:1) gave 0.020 mg (47.5% yield) of titlecompound. ¹H NMR (CDCl₃): δ 8.54 (d, J=5.0 Hz, 2H), 7.76 (s, 1H), 7.48(m, 2H), 7.20 (d, J=5.0 Hz, 2H), 7.08 (t, J=8.5 Hz, 2H), 3.42 (s, 3H)ppm; ¹³C NMR (CDCl₃): δ 164.31, 161.03, 149.79, 148.50, 141.15, 141.13,130.79, 130.28, 130.17, 129.04, 128.99, 122.55, 117.75, 115.63, 115.34,58.95 ppm; MS (M+1)⁺ 302.

To a solution of compound 6 (0.064 g, 0.22 mmol) and NaH (0.016 g, 0.66mmol) in 5 mL of DMF was added a solution of ¹³CH₃I (0.029 g, 0.21 mmol)in 0.5 mL of DMF. The solution was stirred for 2 h and concentrated. Theresultant residue was dissolved in 30 mL of EtOAc and washed with water(5 mL). The organic phase was dried and concentrated. Chromatography(EtOAc/hexane 10:1) gave 0.044 mg (66.2% yield) of title compound. ¹HNMR (CDCl₃): δ 8.42 (d, J=5.3 Hz, 2H), 7.65 (s, 1H), 7.37 (m, 2H), 7.11(d, J=5.3 Hz, 2H), 6.97 (t, J=8.7 Hz, 2H), 3.54, 3.07 (d, J=140 Hz, 3H)ppm; ¹³C NMR (CDCl₃): δ 164.25, 160.97, 149.59, 148.45, 141.21, 130.78,130.23, 130.12, 128.99, 128.95, 122.52, 117.65, 115.57, 115.29, 58.84ppm; MS (M+1)⁺ 303.

Comparison of the NOEs observed between the ligand-probe and PCP withthe NOEs observed between the ligand-probe and TTE0020.003.A09,indicates that addition of the antenna moiety provides informationdiscriminating between the locations of the differing binding sites forsecond ligands.

Example IX

Identifying Ligand Location with a Ligand-Probe Containing an AntennaMoiety

This example demonstrates the use of a specificity ligand-probe havingan antenna moiety capable of detecting a proximal second ligand.

As shown in FIG. 22, a 4-chlorophenol F2 lead fragment containing a ureaantenna moiety was used to probe the core protein of F1 fragment4-fluoro-piridyl-pyrazole core ATP-mimic. The antenna moiety was placedsuch that it can extend from the core structure toward a proximalbinding site. Isolation of the methyl group of the F2 fragment due todistance from the other protons allows direct NOE transfer to beselectively observed by obtaining spectra at relatively short mixingtimes. Furthermore, the absence of vicinal protons minimizes relaxationeffects for the methyl protons, thereby providing a stronger signal.

A sample containing the F1 and F2 fragments together with p38α wasprepared. The sample was screened for proximal ligand interactions by 2DNOESY using 24 micromolar activated human p38 α kinase, in 50 mMpotassium phosphate buffer pH 7.6; 20% H₂O/80% D₂O, ligandconcentrations were 50 micromolar at 277K with a 4 hour acquisitiontime.

As shown in FIG. 22, for the p38α-F1/F2 ternary complex, NOEs wereidentified between the aromatic hydrogen protons of the F1 fragment andthe aliphatic protons of the urea antenna of the F2 fragment.

Throughout this application various publications, patents and patentapplications have been referenced. The disclosures of thesepublications, patents and patent applications in their entireties arehereby incorporated by reference in this application in order to morefully describe the state of the art to which this invention pertains.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

1. A method for obtaining a focused library of candidate bindingcompounds for a protein family, wherein the members of the proteinfamily bind a common ligand, comprising the steps of: (a) providing aligand-probe having an antenna moiety and a ligand moiety, wherein atleast one atom intervenes between the ligand moiety and an NMR visiblenucleus of the antenna moiety, wherein the ligand-probe binds to thecommon ligand binding site of a protein, wherein the protein is a memberof the protein family; (b) providing a sample comprising the protein,the ligand-probe and a second ligand under conditions wherein theligand-probe, the second ligand and the protein form a bound complex;(c) detecting a subset of magnetization transfer signals between theantenna moiety of the ligand-probe and the second ligand in the boundcomplex, wherein said signals are obtained from an isotope-edited NOESYspectrum of said sample, thereby determining that the antenna moiety andsecond ligand are proximal in the bound complex; and (d) obtaining apopulation of candidate binding compounds comprising the ligand-probe,or a fragment thereof that binds to the common ligand binding site ofsaid protein, covalently linked to one of a plurality of homologs ofsaid second ligand, whereby the population contains binding compoundsthat bind to members of the protein family.
 2. The method of claim 1,wherein the antenna moiety comprises an isotope selected from ¹³C, ¹⁵N,¹⁹F, ³¹P and ¹¹³Cd.
 3. The method of claim 1, wherein the ligand probehas a plurality of antenna moieties.
 4. The method of claim 1, whereinstep (d) comprises detecting magnetization transfer between the antennamoieties of the ligand-probe and the second ligand in the bound complex,thereby determining that the antenna moieties and second ligand areproximal in the bound complex.
 5. The method of claim 1, wherein theprotein is deuterium labeled.
 6. The method of claim 1, wherein theligand-probe is identified based on visual inspection of a structuremodel for the binding site of the macromolecule.
 7. The method of claim1, wherein said NOESY spectrum is a 2D NOESY spectrum.
 8. The method ofclaim 1, wherein said isotope-edited NOESY spectrum is a ¹³C-editedNOESY spectrum.
 9. The method of claim 1, wherein the ligand moiety ofsaid ligand-probe comprises a common ligand.
 10. The method of claim 9,wherein step (d) comprises obtaining a population of candidate bindingcompounds comprising the common ligand, or a fragment thereof that bindsto the common ligand binding site of said protein, covalently linked toone of a plurality of homologs of said second ligand.
 11. The method ofclaim 9, wherein candidate binding compounds in the population of step(d) have a covalent linkage between the antenna moiety and a homolog ofsaid second ligand.
 12. The method of claim 1, further comprising a stepof observing competitive binding of a common ligand and the ligand-probeto the protein, thereby determining that the ligand-probe binds to thecommon ligand binding site of the protein.
 13. The method of claim 12,wherein binding of the ligand-probe is identified by a method comprisingmeasuring cross-saturation for a bound complex comprising the firstligand bound to the macromolecule.
 14. The method of claim 13, whereinthe cross-saturation is measured using WaterLOGSY.
 15. The method ofclaim 1, wherein step (c) further comprises identifying an atom of theantenna moiety that is proximal to an atom of the second ligand.
 16. Themethod of claim 15, further comprising determining the distance betweenthe atom of the antenna moiety that is proximal to the atom of thesecond ligand.
 17. The method of claim 1, wherein an inter-liganddistance in the bound complex between the ligand moiety in the commonligand binding site and the bound second ligand is estimated based onthe summation of bond lengths, taking into account bond angles, presentin the antenna moiety plus the NOE estimated distance between the NMRvisible nucleus of the antenna moiety and an atom of the second ligand.18. The method of claim 17, wherein the NOE estimated distance is about6 angstroms or less.
 19. A method for obtaining a focused library ofcandidate binding compounds, wherein the members of the protein familybind a common ligand, comprising the steps of: (a) providing aligand-probe having an antenna moiety and a ligand moiety, wherein atleast one atom intervenes between the ligand moiety and an NMR visiblenucleus of the antenna moiety, wherein the ligand-probe binds to thecommon ligand binding site of a protein, wherein the protein is a memberof the protein family; (b) providing a plurality of samples comprisingthe protein and the ligand-probe under conditions wherein theligand-probe and the protein form a bound complex, wherein the proteinis a member of a family of proteins that bind a common ligand; (c)assaying a population of candidate second ligands for the ability totransfer magnetization to the antenna moiety of the ligand-probe in asample from the plurality, wherein said ability to transfermagnetization is assessed by determining a subset of magnetizationsignals of an isotope-edited NOESY spectrum of said sample; (d)identifying, from the population of candidate second ligands, a secondligand that transfers magnetization to the antenna moiety of theligand-probe, thereby determining that the two ligands are proximal toeach other in a ternary bound complex with the protein; and (e)obtaining a population of candidate binding compounds comprising theligand-probe, or a fragment thereof that binds to the common ligandbinding site of said protein, covalently linked to one of a plurality ofhomologs of said second ligand identified in step (d), whereby thepopulation of candidate binding compounds contains binding compoundsthat bind to members of the protein family.
 20. The method of claim 19,wherein the antenna moiety comprises an isotope selected from ¹³C, ¹⁵N,¹⁹F, ³¹P and ¹¹³Cd.
 21. The method of claim 19, wherein the ligandmoiety of said ligand probe comprises a common ligand.
 22. The method ofclaim 19, wherein step (e) comprises obtaining a population of candidatebinding compounds comprising the common ligand, or a fragment thereofthat binds to the common ligand binding site of said protein, covalentlylinked to one of a plurality of homologs of said second ligand.
 23. Themethod of claim 19, wherein candidate binding compounds in thepopulation of step (e) have a covalent linkage between the antennamoiety and a homolog of said second ligand.
 24. The method of claim 19,wherein the protein is deuterium labeled.
 25. The method of claim 19,wherein the ligand-probe is identified based on visual inspection of astructure model for the binding site of the macromolecule.
 26. Themethod of claim 19, further comprising determining that the secondligand binds to a different location on the protein from the commonligand.
 27. The method of claim 19, wherein said NOESY spectrum is a 2DNOESY spectrum.
 28. The method of claim 19, wherein said isotope-editedNOESY spectrum is a ¹³C-edited NOESY spectrum.
 29. The method of claim19, wherein the ligand probe has a plurality of antenna moieties. 30.The method of claim 29, wherein step (c) comprises assaying a populationof candidate second ligands for the ability to transfer magnetization tothe plurality of antenna moiety of the ligand-probe in a sample from theplurality.
 31. The method of claim 19, further comprising a step ofobserving competitive binding of a common ligand and the ligand-probe tothe protein, thereby determining that the ligand-probe binds to thecommon ligand binding site of the protein.
 32. The method of claim 31,wherein binding of the ligand-probe is identified by a method comprisingmeasuring cross-saturation for a bound complex comprising the firstligand bound to the macromolecule.
 33. The method of claim 32, whereinthe cross-saturation is measured using WaterLOGSY.
 34. The method ofclaim 19, wherein step (d) further comprises identifying an atom of theantenna moiety that is proximal to an atom of the second ligand.
 35. Themethod of claim 34, further comprising determining the distance betweenthe atom of the antenna moiety that is proximal to the atom of thesecond ligand.
 36. The method of claim 19, wherein an inter-liganddistance in the bound complex between the ligand moiety in the commonligand binding site and the bound second ligand is estimated based onthe summation of bond lengths, taking into account bond angles, presentin the antenna moiety plus the NOE estimated distance between the NMRvisible nucleus of the antenna moiety and an atom of the second ligand.37. The method of claim 36, wherein the NOE estimated distance is about6 angstroms or less.